Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR DETECTION OF METASTATIC PROSTATE CANCER
Statement of Government i hts
This invention was made with the support of the U.S.
Government via grants from the National Institutes of Health (Grant Nos.
CA70893 and DK41995). The Government may have certain rights in this
invention.
background of the Invention
The glandular kallikreins are a subgroup of serine proteases which
are involved in the post-translational processing of specific polypeptide
precursors to their biologically active forms. In humans, three members of
this
family have been identified, and some of their properties characterized
(Clements, Endoc. Rev., 1~( , 343 ( 1989); Clements, Mol. Cell Endo., ~9, 1
( 1994); Jones et al., Acta Endoc., 127, 481 ( 1992)). The hKLK 1 gene encodes
the tissue kallikrein protein, hKl, the hKLK2 gene encodes the prostate-
specific
glandular kallikrein protein, hK2, and the hKLK3 gene encodes the prostate-
specific antigen protein, hK3 (PSA). Northern blot analysis of mRNA shows
that both hK2 and PSA are expressed mainly in the human prostate, while
expression of hK 1 is found in the pancreas, submandibular gland, kidney, and
other nonprostate tissues (Chapdelaine et al., FEBS Lett., ?~ø, 205 (1988);
Young et al., Biochem. ~, 818 (1992)).
The nucleotide sequence homology between the exons of hKLK2
and hKLK3 is 80%, whereas the nucleotide sequence homology between the
exons of hKLK2 and hKLKI is 65%. The deduced amino acid sequence
homology of hK2 to PSA is 78%, whereas the deduced amino acid sequence
homology of hK2 to hKl is 57%. Moreover, the deduced amino acid sequence
of hK2 suggests that hK2 may be a trypsin-like protease, whereas PSA is a
chymotrypsin-like protease.
PSA levels are widely used as a prognostic indicator of prostate
carcinoma. However, since the concentration of PSA in serum is elevated in
patients with either prostatic cancer (pCa) or benign prostatic hyperplasia
(BPH),
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the detection of elevated levels of PSA does not distinguish between these
diseases. Moreover, the high degree of homology of hK2 to PSA raises some
question as to the specificity of antibodies currently used to detect the
levels of
PSA. If the levels of circulating hK2 are unrelated to pCa or BPH, then
antibodies raised to preparations of PSA which are contaminated with hK2, or
to
regions of PSA with homology to hK2, can result in false positive results.
Although it is now generally accepted that serum PSA testing,
combined with the digital rectal exam (DRE), is the most effective means to
detect clinically significant and organ-confined prostate cancer, combinations
of
PSA, DRE and ultrasonic prostate examination can detect only some prostate
tumors. For example, up to 40% of surgically treated patients with prostate
cancer are subsequently found to be clinically understaged. Moreover, the
actual
incidence of histological cancers based on autopsy data relative to the
incidence
of clinically significant prostate cancer is high. Furthermore, approximately
30% of patients with alleged localized prostate cancer may have occult
(distant)
metastatic disease (Moreno et al., Cancer Res., 52, 6110 ( 1992)). Of these
patients, 80% experience relapse biochemically, i.e., elevated PSA levels, or
by
the recurrence of local, or occurrence of frank systemic, disease, after
therapy.
Operative therapy is not the appropriate treatment modality for
patients having established metastasis. Screening modalities to assess early
metastases often fail to identify a significant subset of patients with
locally
invasive disease involving penetration of the prostate capsule or seminal
vesicle.
While immunohistochemical techniques have been employed to identify
micrometastatic, or circulating, prostate tumor cells when no obvious
metastatic
deposit was evidenced by conventional means, immunohistochemical methods
are laborious and lack the sensitivity needed for the early detection of
metastatic
or locally invasive prostate cancer.
There is, therefore, a need for early detection of prostate cancer
cells with metastatic potential. Moreover, there is a need to accurately stage
prostate cancer prior to subjecting a patient to invasive procedures. In
particular,
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there is a need for a marker for prostate cancer that can function
independently
of, or in combination with, PSA.
Summary of the Invention
The invention provides a diagnostic method for detecting hK2
DNA wherein the presence of prostate cancer cells in a physiological sample
can
be correlated to the detection of hK2 RNA in the sample. Because expression of
hK2 is prostate tissue specific, hK2 RNA should theoretically not be
detectable
in cells present in bodily fluids or non-prostate tissue if there is no
locally
invasive or metastatic disease, or if all prostate tissue (benign and
malignant) has
been removed or destroyed. The method comprises contacting an amount of
DNA obtained by reverse transcription (RT) of RNA from a human
physiological sample with a plurality of oligonucleotide primers, preferably
at
least two oligonucleotide primers, at least one of which an hK2-specific
oligonucleotide, in an amplification reaction so as to yield an amount of
1 S amplified hK2 DNA. A preferred amplification reaction is a polymerase
chain
reaction (PCR). The presence of the amplified hK2 DNA is then detected. As
described hereinbelow, the presence of amplified hK2 DNA in blood cells, after
RT-PCR, is correlated with prostate cancer, i.e., sixty-seven percent (67%) of
the
prostate cancer patients expressed hK2, 17% expressed PSA,, and 17% expressed
both hK2 and PSA. Preferably, the source of the sample to be tested is human
tissue, e.g., prostate, prostate capsule, seminal vesicle, bone marrow or
lymph
node. Another preferred source of the sample to be tested is a human
physiological fluid which comprises cells, e.g., blood, serum, or seminal
fluid.
As used herein, "amplified hK2 DNA" is defined to mean hK2
DNA in a sample, which was subjected to an amplification reaction, that is
present in an amount that is greater than, i.e., 10, preferably 104, and more
preferably 106, times greater than, the amount of hK2 DNA which was present in
the sample prior to amplification.
As used herein, the term "hK2-specific oligonucleotide" or "hK2-
specific primer" means a DNA sequence that has at least about 80%, more
preferably at least about 90%, and more preferably at least about 95%,
sequence
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identity or homology to SEQ ID N0:4 in regions of SEQ ID N0:4 that are
divergent from nucleotide sequences which encode hK3 (SEQ ID N0:23). An
oligonucleotide or primer of the invention has at least about 7-50, preferably
at
least about 10-40, and more preferably at least about 15-35, nucleotides.
Preferably, the oligonucleotide primers of the invention comprise at least 7
nucleotides at the 3' of the oligonucleotide primer which have at least about
80%,
more preferably at least about 85%, and more preferably at least about 90%,
identity to SEQ ID N0:2, SEQ ID N0:4, or SEQ ID N0:6. The
oligonucleotides of the invention may also include sequences which are
unrelated to hK2 nucleic acid sequences, e.g., they may encode restriction
endonuclease recognition sequences. A preferred hK2-specific oligonucleotide
of the invention comprises SEQ ID N0:14. Another preferred hK2-specific
oligonucleotide of the invention comprises SEQ ID N0:17. Yet another
preferred hK2-specific oligonucleotide of the invention comprises SEQ ID
N0:18.
A preferred diagnostic method of the inventian combines RT-
PCR detection of hK2 transcripts with RT-PCR detection of transcripts of other
gene products associated with prostate cancer. Combined detection of two or
more gene products may provide greater diagnostic certainty or yield more
informative staging information. Combined detection may also be helpful in
differentiating those cells with aggressive growth potential from those that
are
more indolent. In a particularly preferred embodiment of the method provided
by the invention, RT-PCR detection of hK2 RNA is combined with RT-PCR
detection of PSA RNA.
The invention further provides a diagnostic method for detecting
hK2 RNA. The method comprises extracting RNA from a physiological sample
obtained from a human. The extracted RNA is reverse transcribed to yield DNA.
The DNA is contacted with an amount of at least two oligonucleotides effective
to amplify the DNA to yield an amount amplified hK2 DNA, wherein at least
one oligonucleotide is an hK2-specific oligonucleotide. The presence of the
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amplified hK2 DNA is then detected. The presence of the amplified hK2 DNA is
indicative of metastatic prostate cancer in the human.
The presence of hK2 RNA, or a level of hK2 RNA that rises over
time, in bodily fluids or non-prostate tissue may be reasonably expected to
indicate the presence of previously undiagnosed metastatic disease. Early
detection of metastatic disease provides a "lead time" during which
alternative
therapeutic strategies, including those that may not exist at the time of
surgery
but are subsequently developed, can be evaluated. Thus, the present invention
provides a method for monitoring the progression of prostate cancer.
The method comprises contacting an amount of DNA obtained
by reverse transcription of RNA from a physiological sample obtained from a
human afflicted with prostate cancer with an amount of at least two
oligonucleotides, at least one of which an hK2-specific oligonucleotide,
effective
to amplify the DNA to yield an amount of amplified hK2 DNA. The presence or
amount of the amplified hK2 DNA is detected or determined. At least one point
later in time, another sample is taken and the amount of amplified hK2 DNA
detected or determined. Then the amounts of amplified hK2 DNA, obtained at
least at two different time points, are compared.
Also provided is a method for pathologically staging prostate
cancer in a human. The method comprises contacting an amount of DNA
obtained by reverse transcription of RNA from a physiological sample obtained
from the human afflicted with prostate cancer with an amount of at least two
oligonucleotides, at least one of which is an hK2-specific oligonucleotide,
effective to amplify the DNA to yield an amount of amplified hK2 DNA. The
presence or amount of the amplified hK2 DNA is then detected or determined.
The presence or amount of amplified hK2 DNA is indicative of the pathological
stage of the prostate cancer.
Another embodiment of the invention provides a method for
monitoring therapeutic interventions involving hormone therapies. For example,
because hK2 expression is androgen-dependent, hK2 RNA levels in peripheral
blood or other bodily tissue or fluid may be used as a marker during
intermittent
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androgen therapy, or during androgen provocative testing, wherein a patient is
temporarily placed in a hyperandrogenized state to stimulate a level of hK2
production by any persistent prostate cancer cells sufficient to render them
detectable. See T. K. Takayama et al., Sem. in Oncol., 21, 542-553 (1994), and
S references cited therein. hK2 levels are preferably monitored periodically
during
the course of hormone therapy. It may be advantageous to also determine hK2
levels before commencement of therapy, and periodically after the conclusion
of
a therapeutic regimen.
Also provided is a diagnostic kit for detecting hK2 RNA in a
physiological sample suspected of containing hK2 RNA. The kit comprises
packaging containing (a) a known amount of a first hK2-specific
oligonucleotide, wherein the oligonucleotide consists of at least about 7-50
nucleotides, and wherein the oligonucleotide has at least about 80% identity
to
SEQ ID NO: 4, and (b) a known amount of a second hK2-specific
oligonucleotide, wherein the oligonucleotide consists of at least about 7-50
nucleotides, and wherein the oligonucleotide has at least about 80% identity
to a
nucleotide sequence which is complementary to SEQ ID N0:4.
The invention further provides an isolated, purified peptide
comprising SEQ ID N0:22, a biologically active subunit thereof, or a
biologically active variant thereof. The invention fizrther provides an
isolated,
purified peptide comprising SEQ ID N0:26, a biologically active subunit
thereof, or a biologically active variant thereof. Also provided is an
isolated
purified antibody or antibody preparation that specifically reacts with a
protein
or polypeptide which comprises the peptides of the invention described above.
As used herein, the term " biologically active subunit" of a
peptide of the invention is preferably defined to mean a subunit of a peptide
having SEQ ID N0:22, which has at least about 10%, preferably at least about
50%, and more preferably at least about 90%, the activity of a peptide having
SEQ ID N0:22. The activity of a peptide of the invention can be measured by
methods well known to the art including, but not limited to, the ability of
the
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peptide to elicit a sequence-specific immunologic response when the peptide is
administered to an organism, e.g., goat, rabbit, sheep or mice.
As used herein, the term "biologically active variant" of a peptide
of the invention is preferably defined to mean a peptide which has at least
about
80%, preferably at least about 90%, and more preferably at least about 95%,
identity or homology to SEQ ID N0:22. Biologically active variants of the
peptides of the invention have at least about 10%, preferably at least about
50%,
and more preferably at least about 90%, the activity of a peptide having SEQ
ID
N0:22. The activity of a variant peptide of the invention can be measured by
methods described hereinabove.
The invention further provides a method for detecting or
determining the presence of metastatic prostate cancer in a human non-prostate
tissue sample. The method comprises mixing an amount of an agent, which
binds to an hK2 polypeptide and which does not bind to hK3, with the cells of
1 S the mammalian tissue sample so as to form a binary complex comprising the
agent and the cells. The presence or amount of complex formation in the sample
is then detected or determined. The presence or amount of the complex provides
an indication of the presence of micrometastatic prostate cancer. As used
herein, "micrometastatic" means locally invasive disease, which typically
involves penetration of the prostate capsule or seminal vesicle, or occult
disease.
A preferred agent for use in the method is an antibody. The term "antibody"
includes human and animal mAbs, and preparations of polyclonal antibodies, as
well as antibody fragments, synthetic antibodies, including recombinant
antibodies, chimeric antibodies, including humanized antibodies, anti-
idiotopic
antibodies and derivatives thereof. To prepare antibodies which bind to hK2
and
not to hK3, isolated hK2 polypeptides, isolated hK2 peptides, as well as
variants
and subunits thereof, can be used to prepare populations of antibodies. These
antibodies in turn can be used as the basis for direct or competitive assays
to
detect and quantify hK2 polypeptides (or "protein") in samples derived from
tissues such as bone marrow and lymph nodes, and samples of cells such as from
physiological fluids which comprise cells.
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Brief Description of the Figures
Figure 1 depicts the amino acid sequences of wild type mature
hK2 (SEQ ID NO:1) and hK3 (SEQ ID N0:7).
Figure 2 depicts the amino acid sequence, and corresponding
nucleic acid sequence, of wild type pphK2 (SEQ ID N0:3 and SEQ ID N0:4,
respectively), phK2 (SEQ ID NO:S and SEQ ID N0:6) and hK2 (SEQ ID NO:l
and SEQ ID N0:2). Codon 217 (GCT, Ala) is shown in bold and underlined.
Figure 3 is a schematic diagram of the pGT expression vectors
pGThK2 and pGThK2"2".
Figure 4 depicts chromatographic profiles from the purification of
phK2"2". (A) A DEAE chromatogram of 7 day spent medium from AV 12 cells
transfected with a vector encoding pphK2"z". A sample of the spent medium
was applied in bicarbonate buffer, pH 8 and eluted with a salt gradient. The
Azgo
elution profile is represented by a solid line. The dotted line represents the
results of an ELISA assay of a portion of individual column fractions which
was
dried onto microtiter plates and developed with a rabbit anti-pphK2 antibody.
(B) The hydrophobic interaction profile of pooled DEAE fractions. Fractions 24
to 30 from the DEAE chromatographic eluates of (A) were pooled, concentrated
and applied to an HIC (hydrophobic interaction column) column in 1.2 M
sodium sulfate, and eluted with a decreasing salt gradient. The elution
profile
(AZgo) is represented by a solid line. The dotted line represents the results
of an
ELISA assay of a portion of individual column fractions which was dried onto
microtiter plates and developed with a rabbit anti-hK2 antibody. (C) hK2-
containing fractions from the 22 minute peak from (B) were concentrated and
applied to a Pharmacia S 12 size exclusion column. Fractions were collected
and
analyzed by SDS/PAGE. The 19.4 minute peak appeared homogeneous by
SDS-PAGE.
Figure 5 represents an SDS/PAGE analysis of purified hK2 and
PSA. A 1.5 mg sample of purified phK2"2" or PSA was boiled in sample buffer
with (R) or without (N) 1 % beta-mercaptoethanol. Samples were subjected to
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SDS/PAGE on a 4-20% gel. The protein bands were visualized by staining the
geI with silver.
Figure 6 depicts Conconavaiin A staining of phK2°z". The
predicted position of phK2 is designated by an arrow. ZCE (an anti-CEA mAb)
S and B SA were included as examples of glycosylated and non-glycosylated
proteins, respectively. The presence of a band at the predicted position in
the
phK2 lane demonstrates that this protein is glycosylated.
Figure 7 represents the conversion of pro to mature hK2~z" by
trypsin cleavage. Trypsin ( 1 % w/w) was incubated with phK2°2" for 10
minutes
at 37°C in 100 mM borate buffer pH 8, and then subjected to HIC-HPLC.
The
dashed line represents the profile of the phK2"2" prior to incubation with
trypsin.
The solid line represents the profile of phK2 after trypsin digestion. The
profiles
have been superimposed for comparison. The identity of the two forms was
confirmed by N-terminal sequencing of the protein.
Figure 8 depicts Western blot analysis of seminal fluid using
monoclonal antibody (mAb) hKIG 586.1. Processed seminal fluid was diluted
1:1 in PBS and centrifuged at 10,000 X g for 20 minutes. The supernatant was
subjected to SDS/PAGE on a 8-25% gel using the PhastSystem (Phanmacia).
Protein was transferred to nitrocellulose and incubated with protein-G
purified
HK1 G 586.1 ( 1 ~.g/ml) followed by goat anti-mouse IgG-HRP ( 1:1000). The
blot was developed using the ECL detection system (Amersham).
Figure 9 represents a time course study of hK2 expression in
AV 12 cells. AV 12-hK2 clone #27 was grown to ~ 60-70% confluency, then
cells were washed with HBSS and serum free HH4 media was added. Spent
medium was withdrawn each day, concentrated and subjected to SDS/PAGE on
a 12% gel. Proteins were electroblotted and probed with monoclonal antibody
HK1 D 106.4, which detects both phK2 and hK2 ( 1:1000) or HKl G 464.3, which
detects phK2 ( 1:1000), followed by goat anti-mouse IgG-HRP ( 1:500). The blot
was developed with ECL (Amersham) according to the manufacturer's
instructions. Purified phK2°Z" and hK2°2" were used as controls.
The position
of hK2 is indicated by the arrow.
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Figure 10 represents a time course study of expression of the
variant form of hK2 in transfected AV 12 cells. At ~60-70% confluency, AV 12-
hK2"Z" cells were washed with HBSS and serum free HH4 media was added.
Spent media was withdrawn each day, concentrated and subjected to SDS/PAGE
5 on a 12% gel. Proteins were electroblotted and probed with monoclonal
antibodies HK1 D 106.4 and HK1 G 464.3. Goat anti-mouse IgG-HRP { 1:500)
was used as a secondary antibody and the blot was developed with ECL
(Amersham) according to the manufacturer's instructions. Purified phK2"Z" and
hK2"2" were used as controls. The position of hK2 is indicated by the arrow.
10 Figure 11 is a plot of hK2 expression and cell viability over time.
AV 12-hK2 clone #27 was grown to 60-70% confluency, washed with HBSS and
serum free HH4 media was added. Spent media was withdrawn each day and the
hK2 concentration was measured by ELISA using HK1D 106.4 or HK1G 464.3
as a primary antibody, and goat anti-mouse IgG-HRP as a secondary antibody.
The reaction was developed with OPD (Sigma, St. Louis, MO). Viable cells
were enumerated daily using trypan blue dye exclusion.
Figure 12 depicts the expression of hK2 in PC3 and DU145 cells.
PC3 and DU145 cells transfected with pGThK2 were grown to ~60-70%
confluency, washed and resuspended in serum free HH4 media. The spent
medium of pGThK2 transfected DU 145 cells was collected 3 days after
resuspension and the spent medium of pGThK2 transfected PC3 cells was
collected 5 days after resuspension. Spent media were concentrated and
subjected to SDS/PAGE on 12% gels. Proteins were electroblotted and probed
with HK1D 106.4 or HKl G 464.3 as described above. Purified phK2"Z" and
phK2"2" were used as controls. The position of hK2 is indicated by an arrow.
Figure 13 depicts the expression of hK2 by selected hK2-
containing AV 12 clones. Cells from hK2 containing AV 12 clone numbers 10,
27, 31 and 32 were grown to ~60-70% confluency and washed with HBSS, then
serum free HH4 media was added. Spent media was withdrawn 7 days after the
addition of serum free media, concentrated and subjected to SDS/PAGE on a
12% gel. Proteins were electroblotted and probed with HKl D 106.4 or HK 1 G
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464.3. Goat anti-mouse IgG-HRP (1:500) was used as a secondary antibody and
the blot was developed with ECL (Amersham) according to manufacturer's
instructions. Purified phK2"2" and hK2"z" were used as controls. The position
of hK2 is indicated by an arrow.
Figure 14 depicts the expression of phK2"2" in selected AV 12-
hK2"2" clones. Cells from AV 12 clone numbers 2, 3, 4, 45 and 48 were grown
to approximately 60-70% confluency and washed with HBSS, and serum free
HH4 media was added. Spent media was withdrawn 7 days after the addition of
serum free media, concentrated and subjected to SDS/PAGE on a 12% gel.
Proteins were electroblotted and probed with HK 1 D 106.4 or HK 1 G 464.3.
Goat anti-mouse IgG-HRP (1:500) was used as a secondary antibody and the
blot was developed with ECL (Amersham) according to manufacturer's
instructions. Purified phK2"2" and hK2"Z" were used as controls. The position
of hK2 is indicated by an arrow.
Figure 15 depicts the amidolytic specificity of hK2"2", hK2, and
PSA for residues 210-236 of hK2. The synthetic peptide {0.63 mM) was
digested overnight at 37°C with 1 ~g/ml hK2, 40 ~.g/ml hK2"2" or 100
~g/ml
PSA, and the digestion products separated by RP-HPLC. Peaks were normalized
to compare the qualitative aspects of cleavage.
Figure 16 depicts the specificity of hK2 and PSA for different
peptide substrates. Open arrows denote peptide bonds cleaved by PSA; solid
arrows denote bonds cleaved by hK2. Peptide # 1 represents amino acid residues
210-236 of hK2. Peptide #2 represents amino acid residues 1-14 of
angiotensinogen, i.e., the renin substrate tetradecapeptide. Peptide #3
represents
amino acid residues -7 to +7 of phK2. Peptide #4 represents amino acid
residues
41-56 of hK2. Peptide #5 represents the amino acid sequence of the oxidized
beta chain of insulin. Peptide #6 represents amino acid residues 196-213 of
PMSA.
Figure 17 depicts the activation of phK2"2" by hK2 but not
hK2"2". phK2"Z" contains the pro leader peptide sequence VPLIQSR, a
sequence not present in hK2"2". Panel A shows phK2"z" incubated with 1
p i i
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w/w hK2. Panel B is a control with 40% w/w hK2"2" incubated with phK2"Z"
for 6 hours.
Figure 18 depicts Western blot analysis of hK2 incubated with
protease inhibitors. Each sample was separated on a 8-25% gradient SDS-
PAGE, blotted and probed with HK1G586.1. hK2 was incubated for 4 hours at
37°C with the following inhibitors: Lane 1, antichymotrypsin (ACT);
lane 2,
alpha 2-antiplasmin; lane 3, anti-thrombin III; lane 4, alpha 1-protease
inhibitor
(anti-trypsin); lane 5, alpha 2-macroglobulin; lanes 1 and 2 show a covalent
complex of the predicted Mr of 90-100 kD. Serpin inhibitors were employed at
20 ~,M, macroglobulin at 2.8 ~.M, and hK2 at 0.175 uM. Lane 5 shows the
higher Mr complexes representing covalent complex formation of hK2 with
alpha 2-macroglobulin.
Figure 19 depicts complex formation of hK2 in human serum.
Western blots of hK2 and PSA were incubated with human serum. hK2 samples
were probed with HK1G586.1 and PSA samples with PSM773 anti-PSA mAb
Lanes 1-6 contain hK2 samples and lanes 7 and 8 are PSA samples. Lane 1
represents an hK2 control. Lane 2 contains hK2 incubated with ACT for 4
hours. Lane 3 represents a serum control with no added protease. Lane 4
contains hK2 incubated for 15 minutes with serum. Lane 5 contains hK2
incubated with serum for 4 hours. Lane 6 contains hK2 incubated with purified
alpha-2 macroglobulin for 4 hours. Lane 7 contains PSA incubated with serum
for 4 hours. Lane 8 contains PSA incubated with purified alpha-2 macroglobulin
for 4 hours.
Figure 20 depicts immunoreactivity of monoclonal antibody
HKl G 586 in untreated human prostate (n=257).
Figure 21 depicts immunoreactivity of monoclonal antibody
HKl G 586 in androgen deprivation therapy-treated human prostate (n=7).
Figure 22 depicts (A) RT-PCR detection of PSA and hK2
mRNAs in RNA extracted from LNCaP cells diluted in whole human blood, and
{B) RT-PCR detection of PSA and hK2 mRNAs in RNA extracted from whole
human blood of the following patients: patient 17, age 31, male control;
patient
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21, age 58, clinical stage B; patient 24, age 83, known metastatic disease
(D2);
patient 26, age 75, pathological stage C (+margins); patient 28, age 57,
pathological stage C (+margins); patient 49, age 64, pathological stage C
(+seminal vesicles); patient 59, age 31, male control; patient 60, age 73,
known
metastatic disease.
Detailed Description of the Invention
The high degree of amino acid sequence homology of hK2 to
PSA, and the fact that the expression of both hK2 and PSA is essentially
limited
to prostate cells, suggests that measuring the amount or presence of hK2 and
PSA in tissue samples, or measuring the levels of hK2 transcripts in
physiological fluids comprising cells, e.g., blood, or in tissue samples,
e.g.,
lymph node, can be useful in the diagnosis and monitoring of prostatic cancer
(pCa).
Definitions
As used herein, the term "hK2 polypeptide" includes recombinant
pre-pro, pro and mature hK2 polypeptides. A mature hK2 polypeptide having
the amino acid sequence shown in Figure 1 (SEQ ID NO:1 ), as well as "variant"
polypeptides which share at least 90% homology with SEQ ID NO:1 in the
regions which are substantially homologous with hK3, i.e., which regions are
not
identified by bars as shown in Figure 1. The variant hK2 polypeptides of the
invention have at least one amino acid substitution relative to the
corresponding
wild type hK2 polypeptide. A preferred variant hK2 polypeptide comprises SEQ
ID N0:8, i.e., a mature hK2 polypeptide having an alanine to valine
substitution
at amino acid position 217. hK2 polypeptides possess antigenic function in
common with the mature hK2 molecule of Figure 1, in that said polypeptides are
also definable by antibodies which bind specifically thereto, but which do not
cross-react with hK3 (or hKl). Preferably, said antibodies react with
antigenic
sites or epitopes that are also present on the mature hK2 molecule of Figure
1.
Antibodies useful to define common antigenic function are
described in detail in Serial No. 08/096,946, now U.S. Patent No. 5,516,639,
i.e.,
polyclonal antisera prepared in vivo against hK2 subunit 41-56.
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"Isolated hK2 nucleic acid" is RNA or DNA containing greater
than 7, preferably 15, and more preferably 20 or more, sequential nucleotide
bases that are complementary to the non-coding or coding strand of the native
hK2 polypeptide RNA or DNA, or hybridizes to said RNA or DNA and remains
stably bound under stringent conditions. Preferably, the isolated nucleic acid
encodes a biologically active hK2 polypeptide, a variant thereof, or a subunit
thereof. The biological activity of an hK2 polypeptide can be detected by
methods well known to the art including, but not limited to, the ability to
react
with antibodies specific for hK2 polypeptides, the ability to cleave hK2-
specific
substrates (see Example 7), or the ability to bind to serum proteins (see
Example
9). A variant hK2 polypeptide or subunit thereof, or a subunit of an hK2
polypeptide, has at least about 10%, preferably at least about 50%, and more
preferably at least about 90%, the biological activity of an hK2 polypeptide
comprising the amino acid sequence of SEQ ID NO:1.
Thus, the RNA or DNA is isolated in that it is free from at least
one contaminating nucleic acid with which it is normally associated in the
natural source of the nucleic acid and is preferably substantially free of any
other
mammalian RNA or DNA. The phrase "free from at least one contaminating
source nucleic acid with which it is normally associated" includes the case
where
the nucleic acid is reintroduced into the source or natural cell but is in a
different
chromosomal location or is otherwise flanked by nucleic acid sequences not
normally found in the source cell. An example of isolated hK2 nucleic acid is
RNA or DNA that encodes a biologically active hK2 polypeptide sharing at least
90% sequence identity with the hK3-homologous regions of the hK2 peptide of
Figure 1, as described above. The term "isolated, purified" as used with
respect
to an hK2 polypeptide is defined in terms of the methodologies discussed
hereinbelow.
As used herein, the term "recombinant nucleic acid," i.e.,
"recombinant DNA" refers to a nucleic acid, i.e., to DNA that has been derived
or isolated from any appropriate tissue source, that may be subsequently
chemically altered in vitro, and later introduced into target host cells, such
as
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cells derived from animal, plant, insect, yeast, fungal or bacterial sources.
An
example of recombinant DNA "derived" from a source, would be a DNA
sequence that is identified as a useful fragment encoding hK2, or a fragment
or
variant thereof, and which is then chemically synthesized in essentially pure
5 form. An example of such DNA "isolated" from a source would be a useful
DNA sequence that is excised or removed from said source by chemical means,
e.g, by the use of restriction endonucleases, so that it can be further
manipulated,
e.g., amplified, for use in the invention, by the methodology of genetic
engineering.
10 Therefore, "recombinant DNA" includes completely synthetic
DNA sequences, semi-synthetic DNA sequences, DNA sequences isolated from
biological sources, and DNA sequences derived from introduced RNA, as well
as mixtures thereof. Generally, the recombinant DNA sequence is not originally
resident in the genome of the host target cell which is the recipient of the
DNA,
15 or it is resident in the genome but is not expressed, or not highly
expressed.
As used herein, "chimeric" means that a vector comprises DNA
from at least two different species, or comprises DNA from the same species,
which is linked or associated in a manner which does not occur in the "native"
or
wild type of the species.
"Control sequences" is defined to mean DNA sequences
necessary for the expression of an operably linked coding sequence in a
particular host organism. The control sequences that are suitable for
prokaryotic
cells, for example, include a promoter, and optionally an operator sequence,
and
a ribosome binding site. Eukaryotic cells are known to utilize promoters,
polyadenylation signals, and enhancers.
"Operably linked" is defined to mean that the nucleic acids are
placed in a functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably linked to DNA
for a polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or enhancer is operably linked to a
coding sequence if it affects the transcription of the sequence; or a ribosome
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binding site is operably linked to a coding sequence if it is positioned so as
to
facilitate translation. Generally, "operably linked" means that the DNA
sequences being linked are contiguous and, in the case of a secretory leader,
contiguous and in reading phase. However, enhancers do not have to be
contiguous. Linking is accomplished by ligation at convenient restriction
sites.
If such sites do not exist, the synthetic oligonucleotide adaptors or linkers
are
used in accord with conventional practice.
"Southern analysis" or "Southern blotting" is a method by which
the presence of DNA sequences in a restriction endonuclease digest of DNA or
DNA-containing composition is confirmed by hybridization to a known, labeled
oligonucleotide or DNA fragment. Southern analysis typically involves
electrophoretic separation of DNA digests on agarose gels, denaturation of the
DNA after electrophoretic separation, and transfer of the DNA to
nitrocellulose,
nylon, or another suitable membrane support for analysis with a radiolabeled,
biotinylated, or enzyme-labeled probe as described in sections 9.37-9.52 of
Sambrook et al., supra.
"Northern analysis" or "Northern blotting" is a method used to
identify RNA sequences that hybridize to a known probe such as an
oligonucleotide, DNA fragment, cDNA or fragment thereof, or RNA fragment.
The probe is labeled with a radioisotope such as 32P, by biotinylation or with
an
enzyme. The RNA to be analyzed can be usually electrophoretically separated
on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or
other
suitable membrane, and hybridized with the probe, using standard techniques
well known in the art such as those described in sections 7.39-7.52 of
Sambrook
et al., supra.
"Stringent conditions" are those that (1) employ low ionic
strength and high temperature for washing, for example, 0.015 M NaCI/0.0015
M sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50°C, or
(2)
employ a denaturing agent such as formamide during hybridization, e.g., 50%
formamide with 0.1 % bovine serum albumin/0.1 % FicolUO.1
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM
I
,____ _~_-
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NaCI, 75 mM sodium citrate at 42°C. Another example is use of 50%
formamide, S x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5 x Denhardt's solution,
sonicated salmon sperm DNA (50 ~g/ml), 0.1 % SDS, and 10% dextran sulfate at
42°C, with washes at 42°C in 0.2 x SSC and 0.1% SDS. See
Sambrook et al.,
supra, for other examples of stringent conditions.
Expression Cassettes or Expression Vectors
An expression cassette or vector comprising a recombinant DNA
sequence encoding hK2 which is operably linked to a promoter functional in a
host cell, may be circular or linear, double-stranded or single-stranded.
Generally, the expression cassette or vector is in the form of chimeric DNA,
such
as plasmid DNA, that can also contain coding regions flanked by control
sequences which promote the expression of the recombinant DNA present in the
resultant cell line. For example, the expression cassette may itself comprise
a
promoter that is active in mammalian cells, or may utilize a promoter already
present in the genome that is the transformation target. Such promoters
include
the CMV promoter, as well as the SV40 late promoter and retroviral LTRs (long
terminal repeat elements). Aside from recombinant DNA sequences that serve
as transcription units for hK2 or portions thereof, a portion of the
recombinant
DNA may be untranscribed, serving a regulatory or a structural function.
The expression cassettes or expression vectors to be introduced
into the cells further will generally contain either a selectable marker gene
or a
reporter gene or both to facilitate identification and selection of
transformed cells
from the population of cells sought to be transformed. Alternatively, the
selectable marker may be carried on a separate piece of DNA and used in a co-
transformation procedure. Both selectable markers and reporter genes may be
flanked with appropriate regulatory sequences to enable expression in the host
cells. Useful selectable markers are well known in the art and include, for
example, antibiotic and herbicide-resistance genes, such as neo, hpt, dhfr,
bar,
aroA and the like. See also Table 1 of Lundquist et al. (U.S. Patent No.
5,554,798).
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Reporter genes are used for identifying potentially transformed
cells and for evaluating the functionality of regulatory sequences. Reporter
genes which encode for easily assayable proteins are well known in the art. In
general, a reporter gene is a gene which is not present in or expressed by the
recipient organism or tissue and which encodes a protein whose expression is
manifested by some easily detectable property, e.g., enzymatic activity.
Preferred genes include the chloramphenicol acetyl transferase gene (cat) from
Tn9 of E. coli, the beta-glucuronidase gene (gus) of the uidA locus of E.
coli, and
the luciferase gene from firefly Photinus pyral is. Expression of the reporter
gene
is assayed at a suitable time after the DNA has been introduced into the
recipient
cells.
Other elements functional in the host cells, such as introns,
enhancers, polyadenylation sequences and the like, may also be a part of the
recombinant DNA. Such elements may or may not be necessary for the function
of the DNA, but may provide improved expression of the DNA by affecting
transcription, stability of the mRNA, or the like. Such elements may be
included
in the DNA as desired to obtain the optimal performance of the transforming
DNA in the cell.
The general methods for constructing recombinant DNA which
can transform target cells are well known to those skilled in the art, and the
same
compositions and methods of construction may be utilized to produce the DNA
useful herein. For example, J. Sambrook et al., Molecular Clonine: A
Laboratory Manual, Cold Spring Harbor Laboratory Press (2d ed., 1989),
provides suitable methods of construction.
Transformation of Host Cells and Recovery of Recombinant hK2 Polype tn ides
The expression cassette or vector comprising the recombinant
DNA encoding an hK2 polypeptide can be readily introduced into the target
cells
by transfection for example, by the modified calcium phosphate precipitation
procedure of C. Chen et al., Mol. Cell. Biol., 7, 2745 ( 1987). Transfection
can
also be accomplished by lipofectin, using commercially available kits, e.g.,
provided by BRL.
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Suitable host cells for the expression of hK2 polypeptide are
derived from multicellular organisms. Such host cells are capable of complex
processing and glycosylation activities. In principle, any higher eukaryotic
cell
culture can be employed in the practice of the invention, whether from
vertebrate
or invertebrate culture. Examples of invertebrate cells include plant and
insect
cells. Numerous baculoviral strains and variants and corresponding permissive
insect host cells from hosts such as Spodoptera frugiperda (caterpillar),
Aedes
aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster
(fruitfly), and Bombyx mori have been identified. See, e.g., Luckow et al.,
Bio/Technologv, ø, 47 (1988); Miller et al., in Genetic Engin rin , J. K.
Setlow
et al., eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al.,
ur , 31 S, 592 ( 1985). A variety of viral strains for transfection are
publicly
available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5
strain of Bombyx mori NPV, and such viruses may be used, preferably for
transfection of Spodoptera frugiperda cells.
When hK2 polypeptide is expressed in a recombinant cell other
than one of human origin, the hK2 polypeptide is completely free of proteins
or
polypeptides of human origin. However, it is necessary to purify hK2
polypeptide from recombinant cell proteins or polypeptides to obtain
preparations that are substantially homogeneous as to hK2 polypeptide. For
example, the culture medium or lysate can be centrifuged to remove particulate
cell debris. The membrane and soluble protein fractions are then separated.
The
hK2 polypeptide may then be purified from the soluble protein fraction and, if
necessary, from the membrane fraction of the culture lysate. hK2 polypeptide
can then be purified from contaminant soluble proteins and polypeptides by
fractionation on immunoaffinity or ion-exchange columns; ethanol
precipitation;
reverse phase HPLC; chromatography on silica or on an anion-exchange resin
such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
gel filtration using, for example, Sephadex G-75; or ligand affinity
chromatography.
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Once isolated from the resulting transgenic host cells, derivatives
and variants of the hK2 polypeptide can be readily prepared. For example,
amides of the hK2 polypeptides of the present invention may also be prepared
by
techniques well known in the art for converting a carboxylic acid group or
5 precursor, to an amide. A preferred method for amide formation at the C-
terminal carboxyl group is to cleave the polypeptide from a solid support with
an
appropriate amine, or to cleave in the presence of an alcohol, yielding an
ester,
followed by aminoiysis with the desired amine.
Salts of carboxyl groups of the hK2 polypeptide may be prepared
10 in the usual manner by contacting the peptide with one or more equivalents
of a
desired base such as, for example, a metallic hydroxide base, e.g., sodium
hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium
carbonate or sodium bicarbonate; or an amine base such as, for example,
triethylamine, triethanolamine, and the like.
15 N-acyl derivatives of an amino group of the present polypeptides
may be prepared by utilizing an N-acyl protected amino acid for the final
condensation, or by acylating a protected or unprotected peptide. O-acyl
derivatives may be prepared, for example, by acylation of a free hydroxy
peptide
or peptide resin. Either acylation may be carried out using standard acylating
20 reagents such as acyl halides, anhydrides, acyl imidazoles, and the like.
Both N-
and O-acylation may be carried out together, if desired. In addition, the
internal
hK2 amino acid sequence of Figure 1 can be modified by substituting one or two
conservative amino acid substitutions for the positions specified, including
substitutions which utilize the D rather than L form.
Acid addition salts of the polypeptides may be prepared by
contacting the polypeptide with one or more equivalents of the desired
inorganic
or organic acid, such as, for example, hydrochloric acid. Esters of carboxyl
groups of the polypeptides may also be prepared by any of the usual methods
known in the art.
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Variant hK2 Polyp tides
It is envisioned that variant hK2 polypeptides have at least one amino
- acid substitution relative to SEQ ID NO:1, SEQ ID N0:3 or SEQ ID NO:S, e.g.,
SEQ ID N0:8 has an alanine to valine substitution at position 217 relative to
SEQ ID NO:l . In particular, amino acids are substituted in a relatively
conservative manner. Such conservative substitutions are shown in Table 1
under the heading of exemplary substitutions. More preferred substitutions are
under the heading of preferred substitutions. After the substitutions are
introduced, the products are screened for biological activity, e.g., ability
to
generate hK2-specific antibodies or to specifically react with hK2-specific
antibodies, i.e., antibodies that bind to hK2 and not to hK3 (PSA).
n
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TABLE 1
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) Iys; gln; asn lys
Asn (N) gln; his; lys; arg gln
Asp (D) glu glu
Cys (C) ser ser
Gln (Q) asn asn
Glu (E) asp asp
GIy (G) pro pro
His (H) asn; gln; lys; arg arg
Ile (I) leu; val; met; ala; leu
phe
norleucine
Leu (L) norleucine; ile; val;ile
met;
ala; phe
Lys (K) arg; gln; asn arg
Met (M) Ieu; phe; ile leu
Phe (F) Ieu; val; ile; ala leu
Pro (P) gly gly
Ser (S) thr thr
Thr (T) ser ser
Trp (V~ tyr tyr
Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe; leu
ala;
norleucine
Amino acid substitutions falling within the scope of the invention, are, in
general, accomplished by selecting substitutions that do not differ
significantly
in their effect on maintaining (a) the structure of the polypeptide backbone
in the
area of the substitution, for example, as a sheet or helical conformation, (b)
the
charge or hydrophobicity of the molecule at the target site, or (c) the bulk
of the
side chain. Naturally occurnng residues are divided into groups based on
common side-chain properties:
( 1 ) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
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(4) basic: asn, gln, his, lys, arg;
(S) residues.that influence chain orientation: gly, pro; and
. (6) aromatic; trp, tyr, phe.
The invention also envisions hK2 variants with non-conservative
substitutions. Non-conservative substitutions entail exchanging a member of
one of the classes described above for another. Amino acid substitutions are
introduced into the DNA molecules of the invention by methods well known to
the art.
Uses of Recombinant hK2 Polype tides
Once isolated, hK2 polypeptide and its antigenically active
variants, derivatives and fragments thereof can be used in assays for hK2 in
samples derived from biological materials suspected of containing hK2 or anti-
hK2 antibodies, as disclosed in detail in U.S. Patent No. 5,516,639. For
example, the hK2 polypeptide can be labeled with a detectable label, such as
via
one or more radiolabeled peptidyl residues, and can be used to compete with
endogenous hK2 for binding to anti-hK2 antibodies, i.e., as a "capture
antigen"
to bind to anti-hK2 antibodies in a sample of a physiological fluid, via
various
competitive immunoassay format for hK2 which uses anti-hK2 antibodies which
are capable of immobilization is carried out by:
(a) providing an amount of anti-hK2 antibodies which are capable of
attachment to a solid surface;
(b) mixing a physiological sample, which comprises hK2, with a
known amount of hK2 polypeptide which comprises a detectable
label, to produce a mixed sample;
(c) contacting said antibodies with said mixed sample for a sufficient
time to allow immunological reactions to occur between said
antibodies and said hK2 to form an antibody-hK2 complex, and
between said antibodies and said labeled polypeptide to form an
antibody-labeled polypeptide complex;
(d) separating the antibodies which are bound to hK2 and antibodies
bound to the labeled polypeptide from the mixed sample;
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(e) detecting or determining the presence or amount of labeled
polypeptide either bound to the antibodies on the solid surface or
remaining in the mixed sample; and
(f} determining from the result in step (e) the presence or amount of
S said hK2 in said sample.
In another format which can detect endogenous hK2 in a sample
by a competitive inhibition immunoassay, a known amount of anti-hK2 antibody
is added to a sample containing an unknown amount of endogenous hK2. The
known amount is selected to be less than the amount required to complex all of
the hK2 suspected to be present, e.g., that would be present in a sample of
the
same amount of sample material obtained from a patient known to be afflicted
with prostate cancer. Next, a known amount of the hK2 polypeptide of the
invention or a subunit thereof, comprising a detectable label is added. If
endogenous hK2 is present in the sample, fewer antibodies will be available to
bind the labeled hK2 polypeptide, and it will remain free in solution. If no
endogenous hK2 is present, the added labeled polypeptide will complex with the
added anti-hK2 antibodies to form binary complexes. Next, the binary antibody-
antigen complexes are precipitated by an anti-mammal IgG antibody (sheep,
goat, mouse, etc.). The amount of radioactivity or other label in the
precipitate
(a ternary complex) is inversely proportional to the amount of endogenous hK2
that is present in the sample, e.g., a pellet containing reduced amounts of
radioactivity is indicative of the presence of endogenous hK2.
Alternatively to the conventional techniques for preparing
polyclonal antibodies or antisera in laboratory and farm animals, monoclonal
antibodies against hK2 polypeptide can be prepared using known hybridoma cell
culture techniques. In general, this method involves prepared an antibody-
producing fused cell line, e.g., of primary spleen cells fused with a
compatible
continuous line of myeloma cells, and growing the fused cells either in mass
culture or in an animal species from which the myeloma cell line used was
derived or is compatible. Such antibodies offer many advantages in comparison
to those produced by inoculation of animals, as they are highly specific and
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sensitive and relatively "pure" immunochemically. Immunologically active
fragments of the present antibodies are also within the scope of the present
invention, e.g., the flab) fragment, as are partially humanized monoclonal
antibodies.
5 Chimeric and Modified Antibodies
Chimeric antibodies comprise the fusion of the variable domains
from one immunoglobulin to the constant domains from another
immunoglobulin. Usually, the variable domains are derived from an
immunoglobulin gene from a different species, perhaps a human. This
10 technology is well known to the art. See, for example, European Patent
Applications, EP-A-0 125,023 (Cabilly/Genetech) and EP-A-0 120,694 and U.S.
Patent No. 4,816,567, which disclose the preparation of variations of
immunoglobulin-type molecules using recombinant DNA technology.
Another approach to prepare chimeric or modified antibodies is to
15 attach the variable region of a monoclonal antibody to another non-
immunoglobulin molecule, to produce a derivative chimeric molecule (see WO
86/01533, Neuberger and Rabbits/Celltech). A further approach is to prepare a
chimeric immunoglobulin having different specificities in its different
variable
regions (see EP 68763A). Yet another approach is to introduce a mutation in
20 the DNA encoding the monoclonal antibody, so as to alter certain of its
characteristics without changing its essential specificity. This can be
accomplished by site-directed mutagenesis or other techniques known in the
art.
The Winter patent application EP-A-0 239 400 discloses the
preparation of an altered, derivative antibody by replacing the
complementarity
25 determining regions (CDRs) of a variable region of an immunoglobulin with
the
CDRs from an immunoglobulin of different specificity, using recombinant DNA
techniques ("CDR-grafting"). Thus, CDR-grafting enables "humanization" of
antibodies, in combination with the alteration of the framework regions.
Human antibodies can also be prepared by reconstituting the
human immune system in mice lacking their native immune system, then
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immunizing the mice so as to yield human antibodies which are specific for the
immunogen.
A "humanized" antibody containing the CDRs of a rodent
antibody specific for an antigen of interest may be less likely to be
recognized as
foreign by the immune system of a human. It follows that a "humanized"
antibody with the same binding specificity, as e.g., HK16464, may be of
particular use in human therapy and/or diagnostic methods.
The manipulation and/or alteration of any given antibody, or
genes) encoding for the same, to generate a derivative antibody is well known
to
the art.
Detection of hK2-Snecif c Transcripts by Reverse Transcrintase-Polvmerase
hain Reaction lRT-P
To detect hK2 encoding RNA transcripts, RNA is isolated from a
cellular sample suspected of containing hK2 RNA, e.g., total RNA isolated from
human prostate tissue. RNA can be isolated by methods known to the art, e.g.,
using TRIZOLT"' reagent (GIBCO-BRL/Life Technologies, Gaithersburg, MD).
Oligo-dT can be employed as a primer in a reverse transcriptase reaction to
prepare first-strand cDNAs from the isolated RNA. Resultant first-strand
cDNAs are then amplified in PCR reactions.
"Polymerise chain reaction" or "PCR" refers to a procedure or
technique in which amounts of a preselected fragment of nucleic acid, RNA
and/or DNA, are amplified as described in U.S. Patent No. 4,683,195.
Generally, sequence information from the ends of the region of interest or
beyond is employed to design oligonucleotide primers. These primers will be
identical or similar in sequence to opposite strands of the template to be
amplified. PCR can be used to amplify specific RNA sequences, specific DNA
sequences from total genomic DNA, and cDNA transcribed from total cellular
RNA, bacteriophage or plasmid sequences, and the like. See generally Mullis et
al., Coid Siring Harbor S~!mn. Ouant. Biol., 51, 263 {1987); Erlich, ed., PCR
Technolo~v, (Stockton Press, NY, 1989). Thus, amplification of specific
nucleic
acid sequences by PCR relies upon oligonucleotides or "primers" having
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conserved nucleotide sequences, wherein the conserved sequences are deduced
from alignments of related gene or protein sequences, e.g., a sequence
comparison of mammalian hK2 genes. For example, one primer is prepared
which is predicted to anneal to the antisense strand, and another primer
prepared
which is predicted to anneal to the sense strand, of a DNA molecule which
encodes an hK2 polypeptide.
In general, the isolated RNA is combined with a primer in a
reverse transcriptase (RT) reaction to generate single strand cDNAs. Oligo-dT
or random sequence oligonucleotides, as well as sequence specific
oligonucleotides, can be employed as a primer in the RT reaction. See
Sambrook et al., supra. The single strand cDNAs are then amplified with
sequence specific primers to yield an amplified product.
To detect the amplified product, the reaction mixture is typically
subjected to agarose gel electrophoresis or other convenient separation
technique, and the presence or absence of the hK2-specific amplified DNA
detected. Detection of the amplified hK2 DNA may be accomplished by
excising or eluting the fragment from the gel (for example, see Lawn et al.,
Nucleic Acids Res., _9, 6103 ( 1981 ), and Goeddel et al., Nucleic Acids Res.,
8_,
4057 (1980)), cloning the amplified product into a cloning site of a suitable
vector, such as the pCRII vector (Invitrogen), sequencing the cloned
insert and comparing the DNA sequence to the known sequence of hK2.
Alternatively, for example, the hK2 amplified DNA may be detected using
Southern hybridization with an hK2-specific oligonucleotide probe, or
comparing its electrophoretic mobility with DNA standards of known molecular
weight.
The invention will be further described by reference to the
following detailed examples.
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Example 1.
Materials and Methods
Construction of mammalian hK2 expression vectors
A cDNA (approximately 820 by long) encoding the entire
prepro-hK2 (pphK2) (from nucleotide #40 to #858 relative to the start site of
the
pphK2 transcript), as shown in Figure 2, was synthesized from RNA of human
BPH tissue using reverse-transcription polymerase chain reaction (RT-PCR)
technology with a pair of hK2 specific oligonucleotide primers
(5'ACGCGGATCCAGCATGTGGGACCTGGTTCTCT 3'; SEQ ID N0:9 and
5'ACAGCTGCAGTTTACTAGAGGTAGGGGTGGGAC 3'; SEQ ID NO:10).
This cDNA was generated such that 5' and 3' ends (with respect to pphK2 sense
sequence) were bracketed with BamHl and Pstl sequences, respectively. The
cDNA was then purified by agarose gel electrophoresis, and digested with
BamHl and Pstl restriction enzymes. The restricted cDNA was ligated with
BamHl-Pstl digested pVL1393 plasmid vector and transformed into the E. coli
HB 1 O 1 strain. E. coli harboring pphK2 cDNA/pVL 1393 plasmid vector were
selected. The pphK2 containing insert was sequenced. Plasmid pphIC2
cDNA/pVL1393 was mass-produced in E. coli and purified by CsCI gradient
ultra-centrifugation.
Plasmid pphK2/pVL1393 in E. coli HB101 has been deposited in
the American Type Culture Collection, Rockville, MD, USA on May 2, 1994
under the provisions of the Budapest Treaty and has been assigned accession
number ATCC 69614.
A 0.8 kb fragment representing the entire pphK2 coding sequence
(Figure 2) was generated by PCR using primers
A (5'ATATGGATCCATATGTCAGCATGTGGGACCTGGTTCTCTCCA3')
(SEQ ID NO:11 ) and
B (5'ATATGGATCCTCAGGGGTTGGCTGCGATGGT3') (SEQ ID N0:12)
and plasmid pVL1393 containing pphK2 (gift from Dr. Young, Mayo Clinic) as
the template. PCR products were inserted into the TA-cloning vector
(Invitrogen
Corp., San Diego, CA) and the DNA of the entire insert was sequenced.
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To obtain the mammalian hK2 expression vectors, the hK2-
containing inserts were isolated from the corresponding TA clones and inserted
into the Bc 11 site of the plasmid pGT-d (Berg et al., Nucl. Acids Res , ~0,
54-85
( 1992)) (gift from Dr. Brian Grinnell, Lilly) under the control of the GBMT
S promoter. The mammalian expression vectors, PLNS-hK2 and PLNC-hK2 were
obtained by cloning the 0.8 kb wild type hK2 insert from the corresponding TA
vector into the plasmids, pLNSX and pLNCX (Miller et al., ' ch., _9, 980
( 1989)), respectively. The orientation of the insert in all the mammalian
expression vectors was confirmed by DNA sequencing.
Generation of recombinant clones
AV 12-664 (ATCC CRL-9595), a cell line derived from
adenovirus-induced tumors in Syrian hamster, and DU145 cells were cultured in
Dulbecco's modified Eagle's medium (high glucose) supplemented with 10%
fetal bovine serum (D10F). PC3 cells were cultured in Minimal Eagle Medium
containing 10% fetal bovine serum. AV 12 cells were transfected with the hK2
expression vectors using the calcium phosphate method (Maniatis et al., supra
{ 1989)). Three days after transfection cells were resuspended in D 1 OF + 200
nM
methotrexate (MTX). Drug-resistant clonal cell lines were isolated after 2-3
weeks and their spent medium was analyzed by Western blots.. PC3 and DU145
cells were transfected with hK2 mammalian expression vectors using
lipofectamine (Gibco-BRL, Gaithersburg, MD) and clones (PC3-hK2 and
DU 145-hK2) were selected in media containing 400 ~g/ml 6418.
Purification and seauencing of the rn otein
AV 12-hK2 clones were grown in D10F + 200 nM MTX. At
about 60% confluency the cells were washed with Hank's balanced salt solution
and resuspended in serum-free HH4 medium. The spent medium was collected
7 days after the addition of serum-free spent medium and stored at -
20°C. To
purify the protein, the serum-free spent medium was concentrated and exchanged
into 50 mM sodium bicarbonate pH 8. Samples were filtered with 0.2 p filters
and then pumped directly onto a TSK DEAE-SPW HPLC column, 21 mm X
150 mm, at a flow rate of 5 ml/minute. Buffer A contained SO mM Na
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bicarbonate pH 7.9 and Buffer B contained 50 mM Na bicarbonate plus 0.5 M
NaCI pH 7.6. The elution profile was developed with a gradient from 0-50%
Buffer B over 3 5 minutes; 50-100% B from 3 5-40 minutes and isocratic elution
at 100% B for 5 minutes before re-equilibration in Buffer A. The flow rate was
5 5 mL/minute throughout. In the above procedure, borate buffer could replace
bicarbonate buffer with no noticeable difference.
DEAE fractions were assayed for the presence of hK2 by the
dried-down ELISA method (see below) using rabbit anti-pphK2 (Saedi et al.,
Mol. Cell. Endoc., 109, 237 ( 1995)). Fractions with hK2 activity were pooled
10 and concentrated by ultrafiltration with membranes ( 10 kD cut off) to
approximately 5-8 mL. Solid ammonium sulfate was then added to a final
concentration of 1.2 M. This sample was then injected onto a PoIyLC,
polypropyl aspartamide column, 1000 pore size, 4.6 mm X 200 mm, to resolve
proteins by hydrophobic interaction chromatography (HIC). Buffer A was 20
I S mM Na phosphate, 1.2 M Na sulfate pH 6.3 and Buffer B was 50 mM Na
phosphate, 5% 2-propanol, pH 7.4. The elution gradient was 0-20% B over 5
minutes; 20-55% B from 5-20 minutes, isocratic at SS% B from 20-23 minutes,
55-100% B from 23-25 minutes; isocratic at 100% B for 2 minutes before re-
equilibration Buffer A. The flow rate was 1 mL/minute. The HIC peak
20 containing hK2 which eluted at about 50% B was exchanged into 50 mM borate
buffer pH 8 by repeated concentration with Centricon-10 (Amicon) 10 K MW
cutoff ultrafiltration. Purity was assessed by both SDS-PAGE and Western blot
analyses. The extinction coefficient used to estimate hK2°Z"
concentrations was
A28° of 1.84 = 1 mg/ml.
25 In some cases the HIC peak containing hK2 was purified further
by size exclusion chromatography (SEC) on a 10/30 Pharmacia S 12 column. In
this case the HIC peak containing hK2 was concentrated by ultrafiltration as
above to less than 1 mL and then applied to the size exclusion column
equilibrated in 100 mM ammonium acetate pH 7 or sodium borate pH 8. The
30 flow rate was 0.7 mL/minute. The hK2 peak was then concentrated by
ultrafiltration. The peak collected off SEC in ammonium acetate was
lyophilized
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to remove the buffer and then was reconstituted in water. An aliquot of this
sample was hydrolyzed in gaseous 6 N HCl under vacuum for 20 hours at
112°C
then reconstituted in 0.1 N HCl and analyzed on a Hewlett Packard Aminoquant
amino acid analyzer utilizing pre-column derivatization of amino acids with
OPA for primary and FMOC for secondary amines.
A HK1G 586.1 (see below) affinity resin was used to purify hK2
by affinity chromatography. HK1G 586.1 monoclonal antibody (mAb) was
coupled with Pharmacia GammaBind plus Sepharose {cat. no. 17-0886)
according to Schneider (~. Biol. Chem., ~S , 10766 (1982)). Briefly, HK1 G
586.1 mAb and resin were incubated overnight at 4°C with rotation.
Resin was
centrifuged (500 X g for 5 minutes at 4°C) and washed twice with 0.2 M
triethanolamine, pH 8.2. Amine groups were cross-linked in fresh cross linker
solution (25 mM dimethyl pimelimidate dihydrochloride in 0.2 M
triethanolamine, pH 8.2) for 45 minutes at room temperature {22°C). The
resin
was quenched with 20 mM ethanolamine, pH 8.2, for 5 minutes at room
temperature and then washed twice with 1 M NaCI, 0.1 M P04, pH 7Ø The
resin was washed two more times with PBS and stored at 4°C with 0.05%
NaN3
until use.
An Applied Biosystems Model 477a pulsed liquid phase
sequencer was used to sequence the proteins and the peptides. The Model 477a
employs automated Edman degradation chemistry to sequentially release amino
acids from the N-terminus followed by PTH derivatization and chromatography
by reversed-phase HPLC. The peptide samples were applied to the sequencer on
biobrene-treated glass fiber filter supports and whole proteins were applied
either
to biobrene-treated filters or to pre-activated Porton filters (Beckman,
Fullerton,
CA). Samples sequenced off blots were first run as mini-gels on the Novex
system (Novex, San Diego, CA) then transferred to Problot PVDF membrane,
visualized with Commassie blue, the appropriate band cut out and sequenced
directly from the PVDF membrane.
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Monoclonal antibod~production
A/J mice were injected with SO ~1 of phK2 in complete Freund's
adjuvant (CFA) i.p. on day 1, 25 ~g of phK2 in incomplete Freund's adjuvant
(IFA) i.p. on day 14 and 25 pg phK2 in PBS i.p. on day 28. Three days prior to
fusion, mice were boosted with 10 ~g of phK2 in PBS i.v. Mice were sacrificed
and a single cell suspension was prepared from the spleens. Immune B cells
were fused with P3.653 myeloma cells. Cloned hybridomas were screened by
ELISA and selected based on the reactivity of supernatants to
hk2°2" and
phK2°2" and minimal reactivity with PSA. Two clones selected by these
criteria, clones HK1G464 and HK1G586, were subcloned using FACStar plus
cell sorter to deposit single cells onto mouse spleen feeder layers. Subclones
HK1G464.3 and HK1G586.1 were used for further studies.
Another fusion, which employed the same protocol described
above except that the immunogen was hK2°2", alum was used instead of
CFA
and IFA, BALB/c mice were used instead of A/J mice, produced clone
HK 1 H247.
Hybridoma clones HK1G464.3, HK1G586.1, HK1H247,
HK 1 A523 and HKD 106.4 have been deposited with the American Type Culture
Collection, in accord with the Budapest Treaty, and granted Accession Nos:
HB 11983, HB 12026, HB 12162, HB 11876 and HB 11937, respectively.
Polyclonal and monoclonal antibod~production to hK2 peptide immunogens
Sheep and goats were immunized subcutaneously with 100 ~.g of
KLH-conjugated peptide in complete Freund's adjuvant (CFA) and boosted at
three week intervals with 100 wg of peptide in incomplete Freund's adjuvant
(IFA).
For monoclonal antibodies to peptide immunogens, Balb/c mice
were immunized subcutaneously with 100 ~.g of KLH-conjugated peptide in
complete Freund's adjuvant (CFA) and boosted at three week intervals with 100
~g of peptide in incomplete Freund's adjuvant (IFA). Alternatively, A/J mice
were immunized twice intraperitoneally with 50 ~g of KLH-conjugated peptide
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and mice with positive titers were boosted intravenously with 25 p.g of
conjugate.
After the first three immunizations, blood from the animals was
tested for the presence of antibody 6 to 10 days following the immunization.
Peptides were immobilized on 0.25 inch polystyrene beads (Clifton, Clifton
Heights, PA) by incubating 1 pg of peptide conjugated to BSA (bovine serum
albumin) per bead in pH 9.6 carbonate buffer over night at 4°C. The
beads
were washed three times with O.O1M phosphate buffered saline (PBS), pH 7.4
with 0.1 % Tween 20 and blocked with 1 % skim milk plus 1 % BSA. These
beads were incubated for 18 hours with 250 pl of a 1:100, 1:1000 or 1:10,000
dilution of animal sera. Following three washes, 250 pl of rabbit anti-sheep,
anti-mouse or anti-goat antibody conjugated to horseradish peroxidase (Cappel-
Organon Teknica Corporation, Durham, NC) was incubated with each bead for 3
hours on a horizontal incubator at 150 rpm. The enzyme signal was quantified
spectrophotometrically using ortho-phenylene-diamine as a substrate. Non-
immune sera was used as a negative control and immune sera measurements
were expressed as multiples of the control reading.
Lymphocytes from the spleens of mice with positive serum titers
were fused with myeloma cells to produce hybridoma cells. Antibodies produced
by clones of these cells were screened as described above. Positive clones
were
subcloned by limiting dilution and rescreened. Monoclonal hybridomas were
injected into the peritoneal cavities of pristine primed mice to obtain
ascitic
fluid.
To purify monoclonal and polyclonal antisera, the antisera
initially were subjected to an IgG separation by precipitation with saturated
ammonium sulfate and size chromatography using an ultragel ACA-34 column.
The polyclonal antisera was further affinity purified using columns produced
by
cyanogen-bromide coupling of the peptides to Sepharose 4B. The purified
antibody was eluted from the column with acidic PBS (pH 2.45).
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ELISA assays
A dried-down ELISA format was used to measure hK2 in the
serum-free spent medium of the clones and in the fractions collected during
hK2
purification. Microtiter plates (Becton Dickinson Labware, NJ) were coated
with 50 ~1 of spent media or column fractions overnight at 37°C. The
wells
were washed with PBS + 0.1% Tween 20 (PBST) and incubated for one hour
with 50 pl of primary antibodies. The wells were washed again with PBS+T and
incubated for one hour at 37°C with 50 pl of goat anti mouse-IgG or
goat anti
rabbit-IgG Fc antibodies coupled with horseradish peroxidase (1:500, Jackson
Immunosearch Laboratories, Inc., West Grove, PA). The wells were washed
with PBST, incubated with o-phenylenediamine dihydrochloride (OPD, Sigma,
MO) for 5 minutes, and the colorimetric reaction was measured at A49o with an
ELISA reader (Biotek Instruments, Inc., model EL310, VT). All samples were
assayed in duplicate. The serum-free spent medium from AV 12 cells transfected
with vector alone was used as negative control.
Antibodies were tested in a solution-based ELISA format using
biotinylated phK2"z", hK2"z", and PSA. PSA was purified by the method of
Sensabaugh and Blake (J. Urolo~v, 144, 1523 (1990)). Twenty ng of
biotinylated hK2"z" or phK2"z" diluted in 50 p,l Buffer A (8.82 mM citric
acid,
82.1 mM sodium phosphate (dibasic), 10% BSA, 0.1 % mannitol, 0.1 % Nonidet
P-40, pH 7.0) or 0.25 ng biotinylated PSA diluted in 10% horse serum (HS) in
PBS was incubated with 50 pl of hybridoma supernatants, negative control
supernatants (i.e., irrelevant hybridoma supernatant for phK2"z" and hK2"z",
or
20 p.g/ml irrelevant purified mAb in HS for PSA), or positive control
supernatants (i.e., 20 pg/ml purified PSM773 (anti-PSA) mAb in HS for PSA, or
HK 1 D 104 (anti "hK2") hybridoma supernatant for phK2"z" and hK2"z").
HC0514, a mAb against hCG, was used as a negative control in PSA assays, and
ZTG085, a mAb against the tau, was used as a negative control in hK2 assays.
These mixtures of antibodies and antigens were allowed to
incubate for 1 hour with shaking in a streptavidin coated microtiter plate
(Labsystems, Helsinki, Finland). The plate was washed 3 times with 300 pl of
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PBS, 0.1 % Tween-20 (PBST), and incubated with I 00 ~.l of gamma-specific
goat anti mouse IgG-horseradish peroxidase conjugate (Jackson
ImmunoResearch Laboratories, Inc., Westgrove, PA), diluted 1:10,000 in HS,
with shaking for 1 hour. After a second PBST washing, color was developed for
5 30 minutes, with shaking, following the addition of 100 pl of 1 mg/ml o-
phenylenediamine in 50 mM phosphate-citrate buffer, 0.03% sodium perborate,
pH 5.0 (Sigma Chemical, St. Louis, MO). The reaction was quenched by the
addition of 50 X14 N HZS04. The color intensity was determined by measuring
the absorbance at 490 nm and 540 nm using a microtiter plate reader.
10 Absorbances above 2.6 at 490 nm were corrected with 540 nm reading. Sample
values are averages ~ standard deviation of triplicates. Control values are
averages of duplicates.
Western blot assavs
Western blot analyses were performed using standard procedures.
15 Serum-free spent media were concentrated ten fold using Centricon 10
(Amicon,
Inc., Beverly, MA) and subjected to SDS/PAGE using a 12% gel (Bio-Rad, Inc.,
Melville, NY). For analytical purposes, SDS/PAGE was performed on a
Pharmacia PhastSystem using 8-25% gradient gels. After electrophoresis,
proteins were transferred onto nitrocellulose membrane and blocked overnight
at
20 4°C with 2% nonfat dried milk in PBS. Blots were rinsed then
incubated with
primary antibody ( 1:1000 dilution of ascites, or 1 ~,g/ml of purified mAbs or
polyclonal Abs) for 1 hour at 22°C. Blots were then washed and
incubated for
minutes with secondary antibody (Goat anti-mouse-HRP or goat anti-rabbit-
HRP, 1:500, Jackson Immunosearch Laboratories, Inc., West Grove, PA). The
25 immunoreactive bands were detected by developing the blot using DAB (Sigma,
St. Louis, MO) plus HZOz or by using the ECL (Amersham, Buckinghamshire,
England). system according to manufacturer's instructions.
Covalent complex formation
To test for covalent complex formation, 0.175 pM hK2 was
30 incubated with 20 ~M of inhibitor at pH 8 in 100 mM borate buffer.
Inhibitors
tested were 1-antichymotrypsin, 1-antitrypsin, 1-antiplasmin, antithrombin and
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36
2-macroglobulin. To 5 ~,1 of hK2 (10 ~g/ml) was added the calculated ~g of
inhibitor prepared in 100 mM borate buffer and, if needed, each sample brought
up to a total volume of 10 ~l. Samples were incubated for 3 hours at
37°C
whereupon 1.5 ~1 of 7 X PhastSystem SDS sample buffer containing 35% 2-
mercaptoethanol was added and the sample boiled for 3 minutes in a water bath.
Samples were diluted 1/4 in SDS sample buffer prior to application to
SDS/PAGE and Western analyses.
Proteolvsis of Peptide Substrates
To determine the ability of hK2 to cleave peptide substrates,
peptides were dissolved in DMSO at 10 mg/ml then diluted 1:10 into 100 mM
borate buffer pH 8 containing PSA, hK2 or trypsin. Typical experiments were
performed as follows: 1 ~1 of peptide was added to 7 ~I of 100 mM borate
buffer and then 2 ~l of hK2 ( 10 g.g/ml), PSA (500 ~g/ml) or trypsin (0.5
~g/ml)
were added. In general, samples were incubated for 16 hours at 37°C.
Samples were quenched with 100 ~l of 0.2% TFA/water and the
quenched sample was applied directly to a Vydac C-18 reversed-phase column
attached to a BioRad Model 800 HPLC equipped with an AS100 autosampler,
dual 1350 pumps and Biodimension scanning UV-VIS detector. Solvent A was
0.1 % TFA/water and Solvent B was acetonitrile containing 0.1 % TFA. The
sample was applied in 90% solvent A and the gradient developed to 60% solvent
B in 10 minutes. Absorbance was monitored simultaneously at 220 nm and 280
nm.
Peaks collected off HPLC were concentrated by vacuum
centrifugation or lyophilization and then applied to the amino acid sequencer
to
identify individual fragments. In some cases 10 ~l of the quenched sample
mixture was applied directly to the sequencing membrane and, since the
sequence was known, the cleavage sites were determined from the distribution
of
amino acids present in each cycle.
Protease assays using chromogenic substrates
Assays to measure the hydrolysis of para-nitroanilide derivatized
substrates were performed using an HP 8452A UV-VIS spectrophotometer
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equipped with a programmable, thermostated 7-position cell holder. Assays
were performed in 100 mM sodium borate pH 8 incubated at 37°C, the
absorbance increase monitored at 405 nm. Methoxysuccinyl-Arg-Pro-Tyr-para-
nitroanilide (Me0-Suc-R-P-Y-pNA) and H-D-pro-phe-arg-para-nitroanilide (P-
F-R-pNA) were 1 mM in the assay.
An ABI model 431 A peptide synthesizer using standard FastMoc
chemistry was employed to synthesize all of the peptides listed in Figure 16
except #2, angiotensinogen and #S, oxidized beta-chain of insulin which were
obtained from Sigma. The mass of each synthesized peptide was confirmed by
mass spectrometry (University of Michigan, Core Facility) using ES/MS. An
ABI Model 477a sequencer described above was employed to confirm peptide
sequence.
Conversi~~hK2"2" to hK2~2"
Samples of phK2"z" at 100-400 g.g/ml in 50 mM sodium borate
1 S were incubated with 1 % w/w trypsin or hK2 at 37°C. The conversion
of pro to
mature was monitored by dilution of 1-2 ~.g of hK2"Z" starting material into
100 pl HIC Buffer A and resolution of the two forms by HIC-HPLC as described
above. The incubation of hK2"z" with phK2"2" was conducted in the same
manner except that comparable amounts of the two forms were incubated
together as seen in Figure 17B.
Example 2.
Expression and nurificahon of hK2"Z" in mammalian cellc
To express hK2 in mammalian cell lines, a 0.8 kb fragment
encoding the entire coding sequence of hK2 (pphK2) (Figure 2) was amplified
using PCR, subcloned into the vector PCR II (TA) and several clones were
isolated. The nucleotide sequence of the entire pphK2 insert in a few of these
clones was determined to detect any mutations that may have been caused by
PCR amplification.
Two clones, one having a wild type hK2 insert, TA-hK2, and one
having a mutant hK2 insert, TA-hK2"z ", were selected for further analysis. TA-
hK2"2" contains a substitution of T for C at codon 650 of hK2 resulting in a
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conservative substitution of valine (GTT) for alanine (GCT) at amino acid
residue 217 of hK2 (Figure 2). To obtain mammalian expression vectors, pphK2
inserts of TA-hK2 and TA-hK2"2" were subcloned into plasmid PGT-d under the
control of the GBMT promoter resulting in plasmids pGThK2 and pGThK2"2"
(Figure 3). The GBMT promoter is composed of several regulatory sequences
and is activated by the adenovirus E1a proteins) (Berg et al., supra (1992)).
To determine whether the product of the pphK2"2" gene would be
expressed in mammalian cells, the plasmid pGThK2"2" was transfected into
AV 12-664 cells. This cell line is derived from a tumor induced in Syrian
hamster
by adenovirus type 12 and expresses the adenovirus E 1 a protein. The Ela
protein activates the GBMT promoter which results in the expression of the
gene
product under the control of this promoter. After 2-3 weeks, MTX-resistant
clonal cells were isolated and their spent medium were analyzed by Western
blots. Several clones were identified which secreted into the media a
polypeptide
immunoreactive to anti-pphK2 antiserum. One clone (AV 12-pGThK2"2" #2)
was selected for further characterization and protein purification.
To purify hK2 polypeptides, the serum-free spent medium from
AV 12-pGThK2"2" clone #2 was collected after 7 days, concentrated and
subjected to anion exchange chromatography (Figure 4A). The peak of hK2
activity eluted at approximately 0.2 M NaCI as determined by ELISA assays
(dotted line). The ELISA assay correlated well with the appearance of about a
34 kD band of protein seen by SDS/PAGE in the same fractions.
The hK2-positive fractions from the anion exchange column
were collected and subjected to hydrophobic interaction chromatography (HIC)
(Figure 4B). A major portion of the AZBO was not retained on HIC column. The
main peak retained on HIC, which eluted at 22 minutes, also showed the highest
peak of activity by ELISA assay (dotted line, Figure 4C). A major protein band
at about 34 kD was also observed by SDS-PAGE. When the 22 minute peak
from HIC was resolved by SEC, typically about 80-90% of the protein AZgo
eluted at 19.4 minutes, a retention time consistent with a protein of
approximately 34 kD (Figure 4C). The only other protein peak on SEC, eluting
CA 02271770 1999-OS-13
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39
at 16.7 minutes, corresponded to about a 70 kD protein observed in previous
purification steps.
To further identify the purified protein, approximately 2.5 ~.g of
the protein was subjected to automated N-terminal analysis which yielded the
following sequence: Val-Pro-Leu-Ileu-Gln-Ser-Arg-Ileu-Val-Gly-Gly-Trp-Glu-.
No competing sequence was evident from the profile of amino acids released
sequentially by the Edman degradation procedure. By analogy to PSA this
protein is phK2"z", since the known sequence of mature PSA (isolated from
seminal fluid) begins with Ileu-Val-Gly- and pPSA and phK2 have been
postulated to have an extra 7 amino acids at the N-terminus (Figure 2). Amino
acid analysis of this protein yielded an amino acid composition consistent
with
the predicted sequence of phK2"~". This phK2 polypeptide was isolated and
purified in mg quantities.
Exgmple 3.
~haracterizatiQn of nhK2"2" and generation of hK2"2"
To examine the efficiency of the purification scheme employed in
Example 2, 1.5 ~g of purified phK2"2" was subjected to SDS/PAGE in the
presence or absence of beta-mercaptoethanol (BME), and the gel was stained
with silver. Results showed that the phK2"2" in the sample was about 95% pure
(Figure 5). It also showed that phK2"2" migrated at about 30 kD in the absence
of BME, and at about 34 kD in the presence of BME. This pattern is similar to
that observed for the PSA purified from seminal fluid (Figure 5).
The amino acid sequence of hK2, deduced from the cDNA
sequence, shows the presence of one potential N-linked glycosylation site at
residue 78 (N-M-S). To determine if this site is glycosylated, phK2"2" was
subjected to SDS/PAGE, transferred to nitrocellulose paper, reacted with
digoxigenin (DIG)-coupled lectins followed by horseradish peroxidase labeled
anti-DIG.
In Figure 6 (lane 1 ), 2 ug of phK2 was stained with concanavalin
A (Con A) suggesting the presence of two nonsubstituted or 2-O-substituted a-
mannosyl residues in the protein. Lane 2 shows Con A staining of the positive
n
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control glycoprotein, ZCE025 mAB. Both the heavy chains (50 kD) and light
chains (25 kD) of this mAb are known to contain N-linked oligosaccharides with
mannose cores. Lane 3 shows that a nonglycosylated protein (BSA) fails to
react
with the Con A lectin. phK2"2" also reacted with RCA (Gal bl-4GlcNAc
S specificity) and AAA, (a( 1-6) linked fucose specificity). This pattern of
lectin
reactivity is consistent with the presence of complex N-linked
oligosaccharides.
The oligosaccharides on phK2"2" also contain sialic acid since both SNA
(sialic
acid linked a(2-6) to galactose) and MAA (sialic acid linked a(2-6) to
galactose
were reactive with phK2"2".
10 The sequence of the pro region of hK2 is VPLIQSR. An
enzymatic cleavage at the carboxy-terminal end of the arginine in this pro
sequence would convert phK2 to hK2. A mild trypsin digestion was developed
to hydrolyze the peptide bond of purified phK2"2" at this position. phK2"2"
was
incubated with 1 % trypsin and the conversion was monitored by HIC-HPLC
15 (Figure 7). This procedure resulted in a complete conversion of phK2"2" to
hK2"z". The peak designated hK2"2" was N-terminally sequenced and shown to
begin with the sequence, IVGGWE, which is the N-terminus for the mature form
of hK2. No sequence other than the above was detected, demonstrating that this
mild trypsin treatment does not result in any significant level of non-
specific
20 cleavage. SDS/PAGE of trypsin-treated samples showed a small but
discernible
increase in mobility, generally consistent with a minor reduction in mass of
826
daltons, the mass of the pro peptide.
Ezam~le 4.
Generation of hK2-suecific A6s
25 phK2"2" and hK2"2" were used as immunogens to generate mAbs
against hK2. Hybridomas were screened based on high reactivity with hK2"2" or
phK2"2" and minimal reactivity with PSA. Representatives of mAbs obtained
from the hybridomas are shown in Table 2. Immunization with phK2"z" resulted
in mAb HK1G586.1 and HK1G 464.3. HK1G586.i was hK2-specific, since it
30 recognized both phK2"2" and hK2"2" but not PSA. On the other hand,
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41
HK 16464 was phK2-specific, since it only recognized phK2"2" and not hK2"2"
or PSA.
Table 2
Specificity of various mAbs raised to hK2"x" and phK2"x"
A. mAbs raised to phK2"x"
mAbs PSA hK2"2" phK2"z"
Irrelevant 0.245 0.162 0.125
Ab
positive control2.242 ~ 0.06 9.196 8.91 ~ 0.02
HK1G586.1 0.150 ~ 0.004 I 1.154 ~ 0.1810.146 t 0.87
(10 pg/ml)
HK1G464.3 0.143 ~ 0.03 0.245 t 0.02 6.644 t 0.17
(Ascites 1:2000)
B. mAb raised to hK2"x"
mAb tested PSA hK2"2" phK2"z"
Irrelevant 0.157 ~ 0.18 0.132 t 0.01 0.153 ~ 0.01
Ab
Positive control2.768 ~ 0.08 8.342 ~ 1.3 9.673 ~ 0.99
Media only 0.129 ~ 0.02 0.240 ~ 0.02 0.247 ~ 0.01
(neg.
control)
HK1H247 0.157 t 0.01 9.34 t 0.7 0.179 t 0.004
Immunization with hK2"2" resulted in mAb HK1H247. This mAb
was hK2-specific since it recognized only hK2"2" but not phK2"2" or PSA.
These results show that phK2"Z" and hK2"2" are effective as immunogens in
generating mAbs specific for different forms of hK2.
Western blot analysis was used to examine if HK1G586
recognizes hK2 in seminal fluid (Figure 8). hK2-immunoreactive bands at about
22 lcD, 33 kD, and 85 kD were recognized by this mAb. A similar hK2-
immunoreactive pattern in seminal fluid was also recently reported by
Deperthes
et al., Biochem. Bio~h_, . Acta, ~, 311 (1995). This result indicates that a
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42
mAb raised to hK2°z" recognizes native hK2 in seminal fluid. All the
antibodies
raised to hK2°z" or phK2"z" also recognized the corresponding form of
hK2 and
phK2 indicating that hK2 and phK2 are immunologically similar to hK2"2" and
phK2°2", respectively (see below).
To prepare additional anti-hK2 antibodies (Abs), a direct primary
structure comparison between the members of human kallikrein gene family and
computer-aided antigenicity and hydrophobicity analyses was conducted. From
this comparison, several hK2 oligopeptide sequences were selected. The
selected hK2 peptides correspond to mature hK2 amino acid residues 8-26 (SEQ
ID N0:19), 15-26 (SEQ ID N0:26), 41-56 (SEQ ID N0:20), 43-66 (SEQ ID
N0:24), 153-167 (SEQ ID N0:21), 17-71 (SEQ ID N0:22) and 210-235 (SEQ
ID N0:25). The peptide corresponding to amino acids 17-71 was synthesized in
order to increase the likelihood of producing antibodies that recognize the
native
form of hK2. The peptides were synthesized and HPLC purified in the Protein
Core Facility at Mayo Clinic/Foundation. Peptides were conjugated with
keyhole limpet hemocyanin (KLH) and BSA for immunogens and assay
reagents, respectively. Sheep, goat and mice were immunized with KLH-hK2
peptides for polyclonal (sheep and goat, SEQ ID Nos:20 and 21, and SEQ ID
Nos:19, 20, 21, 24, 25 and 26, respectively)) and monoclonal antibody (mice;
SEQ ID Nos:l9, 20, 21, 24, 25 and 26) production. The 17-71 peptide (SEQ ID
N0:22) was oxidized to generate an intramolecular disulfide bond between cys
26 and 42 and used to immunize goats and mice for polyclonal and monoclonal
antibody production, respectively.
The hK2 41-56 Ab from sheep was first purified by hK2 41-56
peptide-affinity column and then used for Western blot analysis. The antibody
recognized recombinant hK2. The detection of hK2 by the hK2 41-56 Ab was
abolished by addition of excess hK2 41-56 peptide but not by PSA 41-56
peptide. Moreover, monoclonal anti-hK2 41-56 peptide antibodies were highly
specific for hK2 protein in Western analysis.
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Anti-hK2 153-167 peptide antiserum (sheep) recognized
recombinant hK2. These results suggested that antibodies to peptides 41-56 and
153-167 react with two distinct epitopes in hK2 polypeptides.
Antisera against hK2 amino acid residues 210-235 showed the
highest immunoreactivity.
A goat antiserum raised against hK2 peptide 17-71, which has
69% homology with corresponding region of PSA, recognized recombinant hK2
protein but not PSA.
Rabbit antiserum to bacterially expressed recombinant hK2
protein, which recognizes both PSA and hK2, detected a doublet protein band in
concentrated LNCaP cell medium from LNCaP cells which were treated with
androgen. In contrast, no immunoreactive protein was detected in LNCaP cell
medium from LNCaP cells which were not treated with androgen. Thus, the
immunoreactive proteins were induced by androgen. Moreover, the upper band
in LNCaP media is a PSA-related protein because a PSA-specific antiserum
(rabbit anti-PSA antisera, which was raised against bacterially expressed
recombinant PSA) detected mainly the upper band. The lower band in LNCaP
media is an hK2-related protein because a mouse monoclonal antibody
(HK1A523) against hK2 41-56 peptide that has monospecificity for hK2
recognizes the lower protein band. These results were confirmed by sequence
analysis of N-terminal amino acids of each protein in the doublet bands.
Immunohistochemistry studies of paraffin-embedded human
prostate tissue sections (see Example 10) which employed a monoclonal
antibody for hK2 peptide 41-56 (HK1A523) showed that hK2, like PSA, is
produced in the epithelia, but not in stroma. Moreover, the immunostaining is
specific for hK2 protein in the prostate, as other tissues tested were
negative for
hK2.
Expression of hK2 in mammalian cells
To express wild type hK2 (hK2) in mammalian cells pGThk2
(Figure 3) was transfected into AV 12 cells. Several clones expressing an hK2
Il
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polypeptide were identified by Western analysis using HK 1 D 106.4 (a hK2-
specific mAb raised to a polypeptide corresponding to amino acid residues 17-
71
of hK2). Clone AV 12-hK2#27 (AV 12-hK2) was selected for further analysis
based on its higher hK2 expression level. Cells transfected with vector alone
(pGTD) showed no reactivity with HK1D 106.4.
ELISA using HK1D 106.4 mAb indicated the presence of about
0.5-1 pg/ml of an hK2 polypeptide in the serum-free spent medium of AV 12-
hK2 at day 7. The same method used in purification of phK2"z" from AV 12-
hK2"z" was used to purify hK2 polypeptides from the day 7 spent medium of
AV 12-hK2. This resulted in low yields of purified hK2 polypeptides which
were unstable to the purification procedures.
hK2 polypeptides were partially purified using the above method,
subjected to SDS/PAGE, electroblotted and subjected to N-terminal amino acid
sequencing. This analysis indicated that the hK2 polypeptide in the spent
medium of AV 12-hK2 at day 7 has the sequence, IVGGWECEK at N-terminus.
No competing sequence was evident from the profile of amino acids released
sequentially by the Edman degradation procedure. By comparison to PSA, this
sequence corresponds to mature hK2 (hK2). Amino acid analysis of this protein
was also consistent with that of hK2.
This finding demonstrates that phK2"z" was predominantly
present in the serum-free spent medium of AV 12-hK2"z" at day 7, whereas
predominantly hK2 was present in the serum-free spent medium of AV 12-hK2 at
day 7. To examine the form of hK2 present in the serum-free medium of AV I2-
hK2 at day 1 this material was partially purified by affinity purification
using
HK1G 586.1 mAbs. The 34 kD protein was transferred onto PVDF and was
subjected to N-terminal analysis, yielding a sequence, VPLIQSRIVGG. No
competing sequence was evident from the profile of amino acids released
sequentially by the Edman degradation procedure. Compared with PSA, this
sequence corresponds to phK2. This suggests that the hK2 polypeptide is
secreted as the pro form by both AV 12-hK2 and AV 12-hk2"z" cells. However,
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while phK2"2" is stable and is not converted to hK2"2", phK2 is unstable and
is
easily converted to hK2 extracellularly.
Example 6.
Biosynthesis of hK2
5 To further study the biosynthesis of hK2 in mammalian cells, a
time course study was conducted where serum-free spent media from AV 12-hK2
clone #27 was collected each day for 8 consecutive days, concentrated and
subjected to SDS/PAGE. The proteins were transferred to nitrocellulose
membrane and probed with either HK1D 106.4 or HK1G 464.3 mAbs
10 (Figure 9). As also shown in Figure 9, HK 1 D 106.4 recognizes both phK2
and
hK2 whereas HK1 G 464.3 recognizes only phK2 as its epitope lies in -7 to +7
region of hK2. Expression of hK2 polypeptides (about 34 kD) peaked by day 3
and plateaued thereafter as detected by HK1D 106.4 mAbs. Two other
immunoreactive bands migrating at about 70 kD and about 90 kD were also
15 detected from day 4 onwards.
On the other hand, when the same samples were blotted and
probed with HKl G 464.3, a gradual reduction in the level of hK2 was detected
by day 4. By day 8, very low levels of hK2 were found in the spent medium.
This result shows that phK2 is being secreted into the media by AV 12-hK2
cells
20 and is gradually converted to hK2 extracellularly. Curiously, the 70 kD and
90
kD bands were not observed with HK1 G 464.3 mAbs indicating that these bands
are either homo-oligomers of hK2 or are hK2 covalently complexed with a yet
unknown protein(s). Even though the identity of these bands is not known at
this
time, they can serve as markers for the presence of hK2 in the spent media. In
25 Figure 9, purified phK2"2" and hK2"2" proteins were used as controls.
To study the biosynthesis of hK2"2" in AV 12 cells a similar time
course study was conducted on AV 12-hK2"2" clone #2. As shown in Figure 10,
expression of hK2"Z" polypeptides peaked by day 3 and did not vary much from
day 4 onwards, as detected by HK1D 106.4 mAbs. Similar results were obtained
30 when the blot was probed with HK 1 G 464.3 mAbs (Figure 10). This indicated
that AV 12-pGThK2"2" clone #2 cells are expressing phK2"2" from day 1
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onwards and that for at least 8 days thereafter, this protein is not converted
to the
mature form. These results are in contrast with those of phK2, which is
converted to hK2 if left in the media for 8 days, indicating that phK2"2" is
stable
in media at 37°C for 8 days.
To study whether extracellular conversion of phK2 to hK2
correlates with the viability of AV 12-hK2 clone #27 cells in culture, clone
#27
cells were counted using trypan blue exclusion. Expression of hK2 in the spent
medium was measured by ELISA using both HK1D 106.4 and HK1G 464.3
mAbs. As shown in Figure 11, the number of viable cells peaked at 3 8 million
in culture by day 3 and gradually decreased thereafter. By day 8, the number
of
viable cells were reduced to less than 10 million. The expression of phK2
(measured by HK1G 464.3) also peaked by day 3 and gradually declined
thereafter.
On the other hand, expression of hK2 (measured by HK1D 106.4)
peaked by day 3 but plateaued thereafter. This result indicates that phK2 is
secreted by AV 12-hK2 cells and a fraction of it is gradually converted
extracellularly to hK2 by day 4. Moreover, it shows that con~rersion of phK2
to
hK2 clearly correlates with a decrease in cell viability, indicating that the
extracellular proteases released by the dying cells may be one of the factors)
responsible for this conversion. Expression of hK2 was highest at the point in
which cells were most viable. A decrease in hK2 paralleled a decrease in cell
viability, suggesting the hK2 is secreted by these cells, as opposed to being
released following cell death and lysis. Also, a rise in hK2 corresponded to a
drop in phK2, indicating that the pro form of hK2 was automatically converted
to the mature form over time.
To examine the biosynthesis of hK2 in prostate carcinoma cells
hK2 was expressed in DU145 and PC3 cell lines. DNA encoding pphK2 was
cloned into plasmids pLNCX and pLNSX (Miller and Rosman, BioTechniques,
7, 980 (1989)), under the control of the CMV and SV40 promoters, respectively.
The resulting plasmids, pLNC-hK2 and pLNS-hK2, respectively, were
transfected into PC3 and DU 145 cells, respectively, and clones were selected
in
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media containing 6418. Clones expressing high levels of hK2 were selected
(PC3-hK2 and DU145-hK2) by ELISA and Western blots.
To assess the level of hK2 and phK2 in the media, serum-free
medium of PC3-hK2 and DU145-hK2 cells were subjected to Western blot
analysis using HK 1 D 106.4 {hK2-specific) and HK 1 G 464.3 (phK2-specific)
mAbs (Figure 12). Results showed that phK2 is present in the spent medium of
both DU 145-hK2 and PC3-hK2. This indicates that in prostate carcinoma cells
hK2 is secreted as phK2 and is converted to the mature form extracellularly.
This finding confirms the results previously obtained with AV 12 cells.
Predominantly phK2 was detected in the spent medium of PC3-hK2 cells even
after 7 days, however, predominately hK2 was present in the serum-free medium
of DU 145-hK2 starting from day 1. This is probably due to abundance of
extracellular proteases in DU 145 spent medium.
To examine whether the above results were limited to just one
clone, 3 other independently isolated clones of AV 12-hK2 and 4 other
independently isolated clones of AV 12-hK2"2" were tested for the expression
of
hK2 polypeptides. Serum-free spent medium of the clones were collected at day
7 and tested for the expression of hK2 by Western blots using HK1D 106.4
(hK2-specific) and HK1G 464 (phK2-specific) mAbs (Figures l3. and 14). In
all of the AV 12-hK2 clones, HK1D 106.4 mAb detected not only the major 34
KD band ("hK2") but also the 70 kD and the 90 kD bands that are indicative of
the presence of hK2 (Figure 13). HK1 G 464.3 detected very low levels of phK2
in all of the AV 12-hK2 clones (Figure 14). This result indicates that
predominantly hK2 is present in the spent medium of all the AV 12-hK2 clones
verifying the biosynthetic mechanism established for AV 12-hK2 #27 clone. The
same analyses were used on AV 12-hK2"2" clones (Figure 14). Results indicated
that only phK2"2" was present in the spent medium of these clones at day 7
verifying our findings with the AV 12-hk2"z" clone.
The above results collectively suggest that hK2 is expressed as
the pro form in mammalian cells and is converted to mature form
extracellularly
by as yet unknown proteases. These results also suggest that phK2 may be
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present in the biological fluids and therefore can be a useful diagnostic
marker
for pCa and BPH.
Exam lp a 7.
Enzymatic activi and specificit~r of hK2 and hK2"Z"
S A small amount of hK2 was purified to sufficient purity to
determine its enzymatic activity and substrate specificity. The general
activity
of hK2 was measured by determining its amidolytic activity chromogenically on
p-nitroanilide derivatives of peptides (Table 3). The p-nitroanilide released
by
proteolytic digestion of these substrates is measured at absorbance AqoS. The
substrate methoxysuccinyl-Arg-Pro-Tyr para-nitroanilide (Me0-Suc-R-P-Y-
pNA) is used to measure chymotrypsin-like proteases which cleave at the
phenylalanine. This substrate has been used previously to measure the activity
of PSA (Christensson et al., Eur. J. Biochem., ~4_, 755 (1990)). The substrate
H-D-Pro-Phe-Arg para-nitroanalide (P-F-R-pNA) is specific for trypsin-like
proteases which cleave at arginine (R).
hK2 was found to have overall activity more than 10 times higher
than hK2"z" on P-F-R-pNA and neither protein showed an ability to hydrolyze
Me0-Suc-R-P-Y-pNA, the chymotrypsin substrate. Other comparable substrates
containing trypsin-like sites for cleavage (lysine, arginine) were also tested
and
hK2 was found to hydrolyze the substrate P-F-R-pNA with the highest rate.
These findings indicate that hK2 has trypsin-like activity.
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Table 3
Amidolytic Activity of hK2, hK2"z", PSA and trypsin on Chromogenic
Substrates
Protease Me0-Suc-R-P-Y-pNA P-F-R-pNA
xmol/min/pg/ml xmol/min/~g/ml
hK2"2" 0 8.7 pmol
hK2 0 4.1 nmol
PSA 13.3 pmol 2.2 pmol
Trypsin 3.8 pmol 25.5 nmol
The specificity of hK2 and hK2"2" was examined in more detail
by the use of peptide substrates together with N-terminal amino acid sequence
analysis to determine which peptide bonds had been hydrolyzed. Figure 15
shows amidolytic activity on the polypeptide
CALPEKPAVYTKVVFIYRKWIKDTIAAN, which has both potential trypsin
and chymotrypsin cleavage sites. hK2"2" cleaved at both a trypsin (R-K) and
chymotrypsin (Y-R) site with the trypsin-like cleavage at a 2:1 ratio over the
chymotrypsin-like cleavage. As a control in these experiments phK2"2" was also
incubated with this peptide and showed no amidolytic activity. hK2 showed
specificity different than hK2"2" towards this peptide substrate. No
chymotrypsin-like specificity was seen for hK2 on this substrate and its
activity
was exclusive for the trypsin-like site (R-K). None of the other lysine (K)
residues in this polypeptide were hydrolyzed indicating that the specificity
of
hK2 was exclusive for the arginine (R) residue.
As a control trypsin was also studied on this substrate and cleaved
all lysine (K) and arginine (R) sites except the K-P bond which is known not
to
be a site suitable for trypsin cleavage. Trypsin cleaved the R-K site of the
210-
236 substrate (peptide #1, Figure 16) at rates approximately 4X faster than
hK2
and about 4000X faster than hK2"Z". No chymotrypsin-like bonds were cleaved
by trypsin. PSA cleaved the Y-R bond primarily. A minor trypsin-like activity
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SO
on the R-K bond was also seen for PSA (Figure 15). This was consistent with
the minor trypsin-like activity previously seen for PSA on the chromogenic
substrate (Table 2).
Several other peptide substrates were also incubated with hK2
and PSA (Figure 16). In all of the peptides tested, hK2 had specificity only
for
selected arginines, and PSA primarily for selected tyrosine (Y), phenylalanine
(F) and leucine (L) residues. Only peptide #1 in Figure 16 was cleaved by
hK2"z" as detailed by the chromatograms in Figure 15.
Example 8.
Activation of_phK2"2" b~
The sequence of peptide #3 in Figure 16 corresponds to amino
acid residue -7 to +7 of phK2. This region contains the pro peptide, VPLIQSR,
which is found as an N-terminal leader peptide in phK2"z". As mentioned
above, hK2 was able to cleave this peptide releasing the propeptide region,
but
hK2"z" was not. To delineate if hK2 can cleave this pro sequence on a native
substrate, its ability to convert phK2"z" to hK2"z" was monitored. phK2"z" was
incubated with 1 % hK2 and the conversion was monitored by the HIC-HPLC
method (Figure 17A). Results showed that hK2 was able to convert
phK2°z" to
hK2"z", albeit at a rate about 30X slower than trypsin. When phK2"z" was
incubated with 40% hK2"z", no difference in the ratios of the two hK2 forms
was detected even after 6 hours (Figure 17B). This corroborated previous
observations with the peptide substrate and showed that, even on a native
substrate, only hK2 and not hK2"z" cleaved the pro region of hK2.
These results collectively demonstrate the stability of phK2"z"
and hK2"z" upon extended incubation. When compared with hK2"z", hK2 was
shown to have a higher proteolytic activity, higher degree of specificity and,
in
particular, to have a specificity for the pro form of hK2 as demonstrated by
activity on the pro peptide in Figure 15 and its activity toward phK2"z" in
Figure
17.
These results demonstrate a significant difference in enzymatic
activity between hK2 and hK2"z" and may help explain the low yields associated
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with attempts to purify hK2 from the medium compared to phK2°z". Highly
purified preparation of hK2 may not be stable due to autolysis as seen for
other
active proteases. These results further suggest that, in addition to
immunological
tests, enzymatic activity on hK2-specific substrates could be used to monitor
the
level of this protein in bodily fluids.
Example 9.
Formation of inhibitor complexes with hK2
PSA has been shown to form complexes with a2 macroglobulin
(MG) and the serine protease inhibitor, antichymotrypsin (ACT). To explore its
complex formation, hK2 was incubated with a series of common proteases
present in human plasma (ACT, a2-antiplasmin, antithrombin III, and a 1-
antitrypsin (Travis and Salvesen, Ann. Rev. Biochem., ~2, 65 5 ( 1983 )) and
the
mixtures were analyzed by Western blot (Figure 18). Any covalent complex of
hK2 with these serpins should result in about a 80-100 kD band on SDS/PAGE
under reducing conditions.
ACT and a2-antiplasmin formed significant complexes with hK2
(Figure 18, lane 1 and 2). Antithrombin III (lane 3) and aI-antitrypsin (al
protease inhibitor, lane 4) formed no detectable complex with hK2. MG, a maj
or
component of blood plasma, also rapidly complexed with hK2 (lane 5). This
complex corresponds to Mr of about 200 kD and 120 kD, which were also
formed when PSA was incubated with purified MG (Figure 18, lane 8, see
below). It was particularly interesting that hK2 did not form complexes with a
1-
antitrypsin, even though this protein inhibits a wide range of trypsin-like
proteases (Loebermann et al., J. Mol. Biol., ~, 531 (1984); Carrell and
Travis,
TIBS, ~Q, 20 (1985)).
It was not surprising that hK2 formed a complex with a2-
antiplasmin since this protein has arginine residues in its inhibitor active
site
(Hunt and Dayhoff, Biochem. Bio~,~v. Res. Co~r_r~., ~S, 864 (1980); Chandra et
al., Biochemistry, ~Z, 5055 (1983); Potempa, et al., i c , 24~,, 699 (1985);
Shieh et al., J. Biol. Chem., ~4, 13420 ( 1989); Mast et al., Biochemistry,
30,
1723 (1991)). However, it was also not expected that hK2 would form a
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complex with ACT, since ACT has a leucine in its inhibitor active site.
Clearly
the structural similarities between PSA and hK2 influence their complex
formation with a common inhibitor even though their proteolytic specificity is
entirely different as demonstrated in Figure 16 and Table 2.
When spiked into human female serum hK2 formed a rapid
complex with MG as detected by Western blot (Figure 18). Lane l and lane 3
are hK2 and serum only controls, respectively. Lane 2 is hK2 incubated with
ACT showing the 90 kD hK2-ACT complex and residual hK2. Lanes 4 and 5
are hK2 spiked into serum for 1 S minutes and 1 hour, respectively. Lane 6 is
hK2 incubated with purified MG for 4 hours. Lane 7 is PSA spiked into serum
for 15 minutes and Lane 8 is PSA incubated with purified MG for 4 hours.
These results show that MG is the major hK2 or PSA complex
formed when hK2 or PSA are spiked into human serum in in vitro experiments.
Since PSA complex with ACT is known to occur in the blood serum of patients
with prostate disease, it is believed that hK2 present in serum would also
form
some level of ACT complex.
Discussion
The in vivo protein processing and secretion mechanisms for PSA
or hK2 are not known. The results presented herein show that phK2 is secreted
by AV 12-hK2, DU 145-hK2, and PC3-hK2 cells, indicating that hK2 is normally
secreted as phK2 and the propeptide is cleaved extracellularly. This suggests
that phK2 exists in biological fluids and thus could be a useful diagnostic
marker
for pCa or BPH.
Both the mutant form of hK2 (hK2~z") and the wild type form of
hK2 were purified from AV 12 cells. hK2 was very unstable to the purification
procedures employed which, as found with other proteases, may be due to its
autocatalytic property, and makes it very difficult to purify hK2 or phK2 in
quantities sufficient for use as immunogens and calibrators. In contrast,
phK2"z s 7
is highly stable and is converted to hK2"z", which was also stable, by trypsin
digestion. Purified phK2''z" and hK2°z" provided immunogens to generate
mAbs specific for hK2 and phK2.
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Ex,~mple 10.
Immunoreactivitv of monoclonal antibody 586 with prostate tissue
Immunohistochemistry of normal prostate tissue with HK1523
showed staining in epithelia but not stroma. Moreover, hK2 expression is
prostate-specific as other tissues, e.g., kidney and pancreas, showed no
staining.
To determine if hK2 is expressed in prostate tissue and, if so, is correlated
with
prostate cancer, 264 radical prostatectomy specimens, of which 257 were from
untreated patients (Figure 20) and 7 were from androgen deprivation therapy
treated patients (Figure 21 ), were analyzed in a comparative study. Each
specimen was analyzed for the cytoplasmic expression of hK2 in areas with
benign epithelium, high grade prostatic intraepithelial neoplasia (PIN) and
adenocarcinoma.
Prostate tissue was weighed, measured in three dimensions and
inked. The apex and base were amputated at a thickness of 4-5 mm and serially
sectioned at 3 mm. The remaining prostate was serially sectioned at 4-5 mm
intervals by knife perpendicular to the long axis of the gland from the apex
of the
prostate to the tip of the seminal vesicles. Transverse sections were prepared
and
stained with hematoxylin and eosin. A single slice of the radical
prostatectomy
of each patient which encompassed cancer and benign tissue was fixed in 10%
neutral buffered formalin and embedded in paraffin by methods well known to
the art.
Tissue sections on slides were deparaffinized by immersion in
xylene and then in 95% ethanol. Endogenous peroxidase activity was blocked
by incubating sections for 10 minutes in methanol/HzOz and then rinsing
sections
in tap water. Sections were then placed in 10 mM citrate buffer, pH 6.0, and
steamed for 30 minutes. Sections were cooled for 5 minutes prior to rinsing in
cold running tap water. Nonspecific protein binding was blocked by incubating
sections for 10 minutes with 5% goat serum. Slides were then gently drained.
Primary antibody, hK1G586 or PSM773 at 0.5 gg/ml, was added
to the sections for 30 minutes at room temperature and then the sections were
rinsed with tap water. Tissue sections were then incubated with biotinylated
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rabbit anti-mouse antibody for one hour and rinsed with water. Sections were
incubated with peroxidase conjugated streptavidin (1:500) for 30 minutes, then
rinsed in tap water. Subsequently, sections were incubated with 3-amino-9-
ethylcarbazole (ABC) chromagen solution for 15 minutes prior to rinsing in tap
water. Sections were then counterstained with mercury-free hematoxylin for one
minute and rinsed for 5 minutes in running water. Slides were mounted with
aqueous mounting media (glycerol gelatin). The percentage of cells staining
was
recorded at 10% increments from 0-100% for benign epithelium, high grade PIN
and adenocarcinoma.
Benign atrophic glands showed the least amount of staining,
particularly in areas of inflammation, in which there was virtually no
immunoreactivity. In hyperplastic acini and benign acini with no evidence of
atrophy, there was moderate to intense immunoreactivity, usually appearing in
a
granular pattern in the secretory luminal cell layer just above the nuclei,
often
extending to the luminal surface. There was no staining of the uroepithelium
of
the urethra or the viva montanum, although the underlying glands often showed
immunoreactivity. Basal cells were usually negative.
Specimens with high grade PIN showed intense immunoreactivity
throughout the cytoplasm and in the cytoplasmic apical blebs in a majority of
cases. There was no apparent difference in the immunoreactivity among the
different patterns of PIN except for the cribiform pattern which was usually
decreased in intensity centrally when compared to the periphery.
Carcinoma specimens showed intense cytoplasmic reactivity in
virtually all cases. Cells with abundant cytoplasmic vacuoles showed less
staining, including signet ring cells and areas of mucin; otherwise the
cytoplasm
was intensely stained. The greatest intensity was observed in the highest
grade
adenocarcinoma (Gleason pattern 4) which showed immunoreactivity in virtually
every case. Foci with cribiform carcinoma were similar to cribiform PIN in
that
there was a greater intensity in the periphery than centrally. The peripheral
edge
and the advancing edge of the carcinomas were always intensely
immunoreactive.
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In seven specimens from patients who had undergone androgen
deprivation therapy, there was little or no immunoreactivity in the majority
of
benign atrophic acini, although PIN and adenocarcinoma occasionally showed
intense cytoplasmic staining.
S A summary of the number of cells staining with monoclonal
antibody HK1G586 in the benign epithelium, high grade PIN and
adenocarcinoma is shown in Table 4. Pair wise analysis, i.e., benign versus
PIN,
benign versus carcinoma, and PIN versus carcinoma, revealed significant
differences for each category (P< 0.001, Spearman Rank Correlation).
10 e4
Immunoreactivitv with hKl 586
can ~andard deviation
Benign epithelium 44.3% 10-90
High grade PIN 69.1 % 20-100
15 Adenocarcinoma 80.0% 20-100
(untreated)
Thus, an increase in cytoplasmic hK2 expression in prostate tissue
is correlated with prostatic neoplasia and prostate cancer. Although the data
20 from prostate obtained from androgen deprivation therapy treated patients
is not
statistically significant due to the small sample size, there is a decrease in
hK2
expression in benign epithelium, high grade PIN and adenocarcinoma in these
patients relative to untreated patients. Thus, an increase in hK2 expression
in
prostate is a novel marker for high grade PIN and prostate cancer.
25 Exam In a 11.
RT-PCR detection of hK2 RNA in prostate cancer cells
Because a large percentage of prostate cancers are understaged, it
is of interest to detect hK2 expressing cells present in tissue biopsies,
e.g.,
prostate capsule, bone marrow or lymph node, or in physiological fluid, e.g.,
30 blood, serum or seminal fluid. Such a detection method preferably can
detect a
single hK2 expressing cell in a large number of non-hK2 expressing cells.
Preferably, the method can detect a single hK2 expressing cell in a sample
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56
comprising at least about 10°, more preferably at least about 106, or
even more
preferably 10', cells. To provide such a sensitive detection method, a reverse
transcriptase-polymerase chain reaction (RT-PCR) specific for hK2 transcripts
is
employed.
S A. LNCaP cell line
To determine the sensitivity of detection of an hK2-specific
transcript by RT-PCR, cells of the human PSA- and hK2-expressing LNCaP cell
line were serially diluted in samples of huffy coat cells. The huffy coat
cells
were isolated from whole blood from normal females and males. Venous blood
{5-7 ml) was collected in citrate-dextrose tubes. Samples were centrifuged at
1000 x g for 15 minutes at 4 ° C. Buffy coat cells were recovered from
the top of
the cell pellet.
The mixture of huffy coat cells and LNCaP cells was centrifuged
at 1,500 rpm for five minutes, and RNA extracted from the pelleted cells. RNA
was isolated by an acidic phenol-chloroform-guanidium thiocyanate method
(Chomczynski et al., Anal. Biochem., 162, 156 ( 1987)). RNA samples were
further extracted with chloroform-butanol {4:1; v/v) to remove residual heme,
which can inhibit both reverse transcription and polymerase chain reactions.
The
isolated RNA was then treated with RNase-free DNase.
To prepare first strand cDNAs, an aliquot containing 1 ~g of total
RNA was added to a reverse transcription reaction containing 100 pmol of a
PSA-specific oligonucleotide primer (5' TCATCTCTGTATCC 3'; SEQ ID
N0:13) or 100 pmol of an hK2-specific oligonucleotide primer (5'
GAGTAAGCTCTA 3'; SEQ ID N0:14), and Moloney murine leukemia virus
reverse transcriptase (GIBCO BRL), and brought to a final volume of 25 gL (50
mM Tris-HCI, pH 8.3, 75 mM KCI, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM
each dNTP, and 800 U Moloney murine leukemia virus reverse tranascriptase).
The reaction was incubated at 42°C for fifteen minutes and the enzyme
was heat
inactivated at 95°C for fifteen seconds.
To amplify PSA first strand cDNAs, 10 ~.1 of the PSA-specific
oligonucleotide primed first strand cDNAs was amplified in a PCR (0.2 mM
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57
each dNTP, 0.5 U AmpliTaq polymerase, 50 mM KC1, 10 mM Tris-HCI, pH 8.3,
1.5 mM MgCl2, 0.1 % (w/v) gelatin) with a PSA-specific primer pair. The PSA
PCR employed 50 pmol of PSA-1 (5' GATGACTCCAGCCACGACCT 3'; SEQ
ID N0:15) and 50 pmol of PSA-2 (5' CACAGACACCCCATCCTATC 3'; SEQ
ID N0:16). To amplify hK2 first strand cDNAs, 10 ~l of the hK2 PSA-specific
oligonucleotide primed first strand cDNAs was amplified in a PCR (0.2 mM
each dNTP, 0.5 U AmpliTaq polymerase, 50 mM KCI, 10 mM Tris-HCI, pH 8.3,
1.5 mM MgClz, 0.1 % (w/v) gelatin) with an hK2-specific primer pair. The hK2
PCR employed 50 pmol of hK2-1 (5' GAGGGTTGTGTACAGTCATGGAT 3';
SEQ ID N0:17) and 50 pmol of hK2-2 (5'
ACACACTGAAGACTCCTGGGGCG 3'; SEQ ID N0:18)).
The cycling parameters employed were: thirty-five to forty cycles
of 94°C for 1 minute; 58°C (PSA) or 60°C (hK2) for 90
seconds, and 72°C for
90 seconds. The final cycle was at 72°C for ten minutes. Aliquots of
the
reaction were electrophoresed on 1.0% agarose gels The gels were stained with
ethidium bromide and were viewed under ultraviolet light. Some of the
amplified products were excised from the gel and subcloned into a pCRII vector
(Invitrogen, San Diego, CA) for sequencing.
The PSA-specific PCR yielded a 710 by product while the hK2-
specific PCR yielded a 405 by product. The results of the dilution analysis
showed that PSA and hK2 RNA was detectable at approximately 1 LNCaP cell
in 106 and 10' white blood cells, respectively (Figure 22A). This result was
unexpected because the RT-PCR detected LNCaP-derived hK2 transcripts at a
ten fold high dilution than LNCaP-derived PSA transcripts.
B. Prostate cancer an tients
The blood from six patients with prostate cancer and from two
normal males was analyzed by RT-PCR. Buffy coat cells and isolated RNA
were obtained from all eight males, and RT-PCR was performed, as described
above. The six prostate cancer patients included one with clinical stage B
prostate cancer, two with known metastatic disease (clinical stage D2) and
three
with pathological stage C. Prostate cancer pathological stages A-C are
localized
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forms of prostate cancer. Pathological stage D 1 is prostate cancer which has
spread to the nodes (nodal metastases). Pathological stage D2 is systemic
(systemic metastases) prostate cancer. For a further description of the
pathological stages of prostate cancer, see Moreno et al. (Cancer Res., 52,
6110
(1992)) Deguchi et al. (Cancer Res., 53, 5350 (1993)) and Katz et al.
( rol , ~, 765 (1994)).
The results showed that 67% of the prostate cancer patients
expressed hK2, 17% expressed PSA, and 17% expressed both hK2 and PSA. No
detectable levels of PSA or hK2 RNA were found in normal controls.
Thus, the detection of hK2 RNA may serve as a useful marker for
early detection of micrometastasis of prostate cancer.
The invention is not limited to the exact details shown and
described, for it should be understood that many variations and modifications
may be made while remaining within the spirit and scope of the invention
1 S defined by the claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: Mayo Foundation for Medical Education and Research,
and Hybritech Incorporated
(ii) TITLE OF THE INVENTION: METHOD FOR DETECTION OF METASTATIC
PROSTATE CANCER
(iii) NUMBER OF SEQUENCES: 26
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Schwegman, Lundberg, Woessner & Kluth, P.A.
(B) STREET: P.O. Box 2938
(C) CITY: Minneapolis
(D) STATE: MN
(E) COUNTRY: U.S.A
(F) ZIP: 55402
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: Unknown
(B) FILING DATE: November 14, 1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:08/759,354
(B) FILING DATE:14-NOV-1996
(A) APPLICATION NUMBER: PCT/US96/06167
(B) FILING DATE: 02-MAY-1996
(A) APPLICATION NUMBER: 08/622,046
(B) FILING DATE: 26-MAR-1996
(A) APPLICATION NUMBER: 08/427,707
(B) FILING DATE: 02-MAY-1995
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Embretson, Janet E
(B) REGISTRATION NUMBER: 39,665
(C) REFERENCE/DOCKET NUMBER: 545.005W01
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 612-359-3260
(B) TELEFAX: 612-359-3263
(C) TELEX:
II
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(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 237 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val
1 5 10 15
Ala Val Tyr Ser His Gly Trp Ala His Cys Gly Gly Val Leu Val His
20 25 30
Pro Gln Trp Val Leu Thr Ala Ala His Cys Leu Lys Lys Asn Ser Gln
35 40 45
Val Trp Leu Gly Arg His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gln
50 55 60
Arg Val Pro Val Ser His Ser Phe Pro His Pro Leu Tyr Asn Met Ser
70 75 80
Leu Leu Lys His Gln Ser Leu Arg Pro Asp Glu Asp Ser Ser His Asp
85 90 95
Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Lys Ile Thr Asp Val Val
100 105 110
Lys Val Leu Gly Leu Pro Thr Gln G1u Pro Ala Leu Gly Thr Thr Cys
115 120 125
Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Glu Phe Leu Arg Pro
130 135 140
Arg Ser Leu Gln Cys Val Ser Leu His Leu Leu Ser Asn Asp Met Cys
145 150 155 160
Ala Arg Ala Tyr Ser Glu Lys Val Thr Glu Phe Met Leu Cys Ala Gly
165 170 175
Leu Trp Thr Gly Gly Lys Asp Thr Cys Gly Gly Asp Ser Gly Gly Pro
180 185 190
Leu Val Cys Asn Gly Val Leu Gln Gly Ile Thr Ser Trp Gly Pro Glu
195 200 205
Pro Cys Ala Leu Pro Glu Lys Pro Ala Val Tyr Thr Lys Val Val His
210 215 220
Tyr Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala Asn Pro
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225 230 235
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE
CHARACTERISTICS:
(A) 11 pairs
LENGTH: base
7
(B) nucleicacid
TYPE:
(C) double
STRANDEDNESS:
(D) linear
TOPOLOGY:
(ii)MOLECULE cDNA
TYPE:
(xi)SEQUENCE SEQID
DESCRIPTION: N0:2:
ATT GTG GGAGGC TGGGAGTGT GAGAAGCATTCC CAACCCTGG CAGGTG 48
Ile Val GlyGly TrpGluCys GluLysHisSer GlnProTrp GlnVal
1 5 10 15
GCT GTG TACAGT CATGGATGG GCACACTGTGGG GGTGTCCTG GTGCAC 96
Ala Val TyrSer HisGlyTrp AlaHisCysGly GlyValLeu ValHis
20 25 30
CCC CAG TGGGTG CTCACAGCT GCCCATTGCCTA AAGAAGAAT AGCCAG 144
Pro Gln TrpVal LeuThrAla AlaHisCysLeu LysLysAsn SerGln
35 40 45
GTC TGG CTGGGT CGGCACAAC CTGTTTGAGCCT GAAGACACA GGCCAG 192
Val Trp LeuGly ArgHisAsn LeuPheGluPro GluAspThr GlyGln
50 55 60
AGG GTC CCTGTC AGCCACAGC TTCCCACACCCG CTCTACAAT ATGAGC 240
Arg Val ProVal SerHisSer PheProHisPro LeuTyrAsn MetSer
65 70 75 80
CTT CTG AAGCAT CAAAGCCTT AGACCAGATGAA GACTCCAGC CATGAC 288
Leu Leu LysHis GlnSerLeu ArgProAspG1u AspSerSer HisAsp
85 90 95
CTC ATG CTGCTC CGCCTGTCA GAGCCTGCCAAG ATCACAGAT GTTGTG 336
Leu Met LeuLeu ArgLeuSer GluProAlaLys IleThrAsp ValVal
100 105 110
AAG GTC CTGGGC CTGCCCACC CAGGAGCCAGCA CTGGGGACC ACCTGC 384
Lys Val LeuGly LeuProThr GlnGluProAla LeuGlyThr ThrCys
115 120 125
TAC GCC TCAGGC TGGGGCAGC ATCGAACCAGAG GAGTTCTTG CGCCCC 432
Tyr Ala SerGly TrpGlySer IleGluProGlu GluPheLeu ArgPro
130 135 140
AGG AGT CTTCAG TGTGTGAGC CTCCATCTCCTG TCCAATGAC ATGTGT 480
Arg Ser LeuGln CysValSer LeuHisLeuLeu SerAsnAsp MetCys
145 150 155 160
II
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GCT AGA GCT TAC TCT GAG AAG GTG ACA GAG TTC ATG TTG TGT GCT GGG 528
Ala Arg Ala Tyr Ser Glu Lys Val Thr Glu Phe Met Leu Cys Ala Gly
165 170 175
CTC TGG ACA GGT GGT AAA GAC ACT TGT GGG GGT GAT TCT GGG GGT CCA 576
Leu Trp Thr Gly Gly Lys Asp Thr Cys Gly Gly Asp Ser Gly Gly Pro
180 185 190
CTT GTC TGT AAT GGT GTG CTT CAA GGT ATC ACA TCA TGG GGC CCT GAG 624
Leu Val Cys Asn Gly Val Leu Gln Gly Ile Thr Ser Trp Gly Pro Glu
195 200 205
CCA TGT GCC CTG CCT GAA AAG CCT GCT GTG TAC ACC AAG GTG GTG CAT 672
Pro Cys Ala Leu Pro Glu Lys Pro Ala Val Tyr Thr Lys Val Val His
210 215 220
TAC CGG AAG TGG ATC AAG GAC ACC ATC GCA GCC AAC CCC 711
Tyr Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala Asn Pro
225 230 235
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 261 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Met Trp Asp Leu Val Leu Ser Ile Ala Leu Ser Val Gly Cys Thr Gly
1 5 10 .15.
Ala Val Pro Leu Ile Gln Ser Arg Ile Val Gly Gly Trp Glu Cys Glu
20 25 30
Lys His Ser Gln Pro Trp Gln Val Ala Val Tyr Ser His Gly Trp Ala
35 40 45
His Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala
50 55 60
His Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu
65 70 75 80
Phe Glu Pro Glu Asp Thr Gly Gln Arg Val Pro Val Ser His Ser Phe
85 90 95
Pro His Pro Leu Tyr Asn Met Ser Leu Leu Lys His Gln Ser Leu Arg
100 105 110
Pro Asp Glu Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu
115 120 125
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63
Pro Ala Lys Ile Thr Asp Val Val Lys Val Leu Gly Leu Pro Thr Gln
130 135 140
Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile
145 150 155 160
Glu Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gln Cys Val Ser Leu
165 170 175
His Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys Val
180 185 190
Thr Glu Phe Met Leu Cys Ala Gly Leu Trp Thr Gly Gly Lys Asp Thr
195 200 205
Cys Gly Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln
210 215 220
Gly Ile Thr Ser Trp Gly Pro Glu Pro Cys Ala Leu Pro Glu Lys Pro
225 230 235 240
Ala Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr
245 250 255
Ile Ala Ala Asn Pro
260
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 832 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GGATCCAGC ATG TGG GAC CTG GTT CTC TCC ATC GCC TTG TCT GTG GGG 48
Met Trp Asp Leu Val Leu Ser Ile Ala Leu Ser Val Gly
1 5 10
TGC ACT GGT GCC GTG CCC CTC ATC CAG TCT CGG ATT GTG GGA GGC TGG 96
Cys Thr Gly Ala Val Pro Leu Ile Gln Ser Arg Ile Val Gly Gly Trp
15 20 25
GAG TGT GAG AAG CAT TCC CAA CCC TGG CAG GTG GCT GTG TAC AGT CAT 144
Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val Ala Val Tyr Ser His
30 35 40 45
GGA TGG GCA CAC TGT GGG GGT GTC CTG GTG CAC CCC CAG TGG GTG CTC 192
Gly Trp Ala His Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu
50 55 60
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64
ACA GCT GCC CAT TGC CTA AAG AAG AAT AGC CAG GTC TGG CTG GGT CGG 240
Thr Ala Ala His Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg
65 70 75
CAC AAC CTG TTT GAG CCT GAA GAC ACA GGC CAG AGG GTC CCT GTC AGC 288
His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gln Arg Val Pro Val Ser
86 85 90
CAC AGC TTC CCA CAC CCG CTC TAC AAT ATG AGC CTT CTG AAG CAT CAA 336
His Ser Phe Pro His Pro Leu Tyr Asn Met Ser Leu Leu Lys His Gln
95 100 105
AGC CTT AGA CCA GAT GAA GAC TCC AGC CAT GAC CTC ATG CTG CTC CGC 384
Ser Leu Arg Pro Asp Glu Asp Ser Ser His Asp Leu Met Leu Leu Arg
110 115 120 125
CTG TCA GAG CCT GCC AAG ATC ACA GAT GTT GTG AAG GTC CTG GGC CTG 432
Leu Ser Glu Pro Ala Lys Ile Thr Asp Val Val Lys Val Leu Gly Leu
130 135 140
CCC ACC CAG GAG CCA GCA CTG GGG ACC ACC TGC TAC GCC TCA GGC TGG 480
Pro Thr Gln Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp
145 150 155
GGC AGC ATC GAA CCA GAG GAG TTC TTG CGC CCC AGG AGT CTT CAG TGT 528
Gly Ser Ile Glu Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gln Cys
160 165 170
GTG AGC CTC CAT CTC CTG TCC AAT GAC ATG TGT GCT AGA GCT TAC TCT 576
Val Ser Leu His Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Tyr Ser
175 180 185
GAG AAG GTG ACA GAG TTC ATG TTG TGT GCT GGG CTC TGG ACA GGT GGT 624
Glu Lys Val Thr Glu Phe Met Leu Cys Ala Gly Leu Trp Thr Gly Gly
190 195 200 205
AAA GAC ACT TGT GGG GGT GAT TCT GGG GGT CCA CTT GTC TGT AAT GGT 672
Lys Asp Thr Cys Gly Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly
210 215 220
GTG CTT CAA GGT ATC ACA TCA TGG GGC CCT GAG CCA TGT GCC CTG CCT 720
Val Leu Gln Gly Ile Thr Ser Trp Gly Pro Glu Pro Cys Ala Leu Pro
225 230 235
GAA AAG CCT GCT GTG TAC ACC AAG GTG GTG CAT TAC CGG AAG TGG ATC 768
Glu Lys Pro Ala Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile
240 245 250
AAG GAC ACC ATC GCA GCC AAC CCC TGAGTGCCCC TGTCCCACCC CTACCTCTAG 822
Lys Asp Thr Ile Ala Ala Asn Pro
255 260
TAAACTGCAG 832
(2) INFORMATION FOR SEQ ID N0:5:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 244 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Val Pro Leu Ile Gln Ser Arg Ile Val Gly Gly Trp Glu Cys Glu Lys
1 5 10 15
His Ser Gln Pro Trp Gln Val Ala Val Tyr Ser His Gly Trp Ala His
20 25 30
Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala His
35 40 45
Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu Phe
50 55 60
Glu Pro Glu Asp Thr Gly Gln Arg Val Pro Val Ser His Ser Phe Pro
65 70 75 80
His Pro Leu Tyr Asn Met Ser Leu Leu Lys His Gln Ser Leu Arg Pro
85 90 95
Asp Glu Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu Pro
100 105 110
Ala Lys Ile Thr Asp Val Val Lys Val Leu Gly Leu Pro Thr Gln Glu
115 120 125
Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile Glu
130 135 140
Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gln Cys Val Ser Leu His
145 150 155 160
Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys Val Thr
165 170 175
Glu Phe Met Leu Cys Ala Gly Leu Trp Thr Gly Gly Lys Asp Thr Cys
i80 185 190
Gly Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln Gly
195 200 205
Ile Thr Ser Trp Gly Pro Glu Pro Cys Ala Leu Pro Glu Lys Pro Ala
210 215 220
Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr Ile
225 230 235 240
Ala Ala Asn Pro
II
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b6
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 766 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..732
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
GTG CCC CTC ATC CAG TCT CGG ATT GTG GGA GGC TGG GAG TGT GAG AAG 48
Val Pro Leu Ile Gln Ser Arg Ile VaI Gly Gly Trp Glu Cys Glu Lys
1 5 10 15
CAT TCC CAA CCC TGG CAG GTG GCT GTG TAC AGT CAT GGA TGG GCA CAC 96
His Ser Gln Pro Trp Gln Val Ala Val Tyr Ser His Gly Trp Ala His
20 25 30
TGT GGG GGT GTC CTG GTG CAC CCC CAG TGG GTG CTC ACA GCT GCC CAT 144
Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala His
35 40 45
TGC CTA AAG AAG AAT AGC CAG GTC TGG CTG GGT CGG CAC AAC CTG TTT 192
Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu Phe
50 55 60
GAG CCT GAA GAC ACA GGC CAG AGG GTC CCT GTC AGC CAC AGC TTC CCA 240
Glu Pro Glu Asp Thr Gly Gln Arg Val Pro Val Ser His Ser Phe Pro
65 70 75 80
CAC CCG CTC TAC AAT ATG AGC CTT CTG AAG CAT CAA AGC CTT AGA CCA 288
His Pro Leu Tyr Asn Met Ser Leu Leu Lys His Gln Ser Leu Arg Pro
85 90 95
GAT GAA GAC TCC AGC CAT GAC CTC ATG CTG CTC CGC CTG TCA GAG CCT 336
Asp Glu Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu Pro
100 105 110
GCC AAG ATC ACA GAT GTT GTG AAG GTC CTG GGC CTG CCC ACC CAG GAG 384
Ala Lys Ile Thr Asp Val Val Lys Val Leu Gly Leu Pro Thr Gln Glu
115 120 125
CCA GCA CTG GGG ACC ACC TGC TAC GCC TCA GGC TGG GGC AGC ATC GAA 432
Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile Glu
130 135 140
CCA GAG GAG TTC TTG CGC CCC AGG AGT CTT CAG TGT GTG AGC CTC CAT 480
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Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gln Cys Val Ser Leu His
145 150 155 160
CTC CTG TCC AAT GAC ATG TGT GCT AGA GCT TAC TCT GAG AAG GTG ACA 528
Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys Val Thr
165 170 175
GAG TTC ATG TTG TGT GCT GGG CTC TGG ACA GGT GGT AAA GAC ACT TGT 576
Glu Phe Met Leu Cys Ala Gly Leu Trp Thr Gly Gly Lys Asp Thr Cys
180 185 190
GGG GGT GAT TCT GGG GGT CCA CTT GTC TGT AAT GGT GTG CTT CAA GGT 624
Gly Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln Gly
195 200 205
ATC ACA TCA TGG GGC CCT GAG CCA TGT GCC CTG CCT GAA AAG CCT GCT 672
Ile Thr Ser Trp Gly Pro Glu Pro Cys Ala Leu Pro Glu Lys Pro Ala
210 215 220
GTG TAC ACC AAG GTG GTG CAT TAC CGG AAG TGG ATC AAG GAC ACC ATC 720
Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr Ile
225 230 235 240
GCA GCC AAC CCC TGAGTGCCCC TGTCCCACCC CTACCTCTAG TAAA 766
Ala Ala Asn Pro
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 237 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Ile Val Gly G1y Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val
1 5 10 15
Leu Val Ala Ser Arg Gly Arg Ala Val Cys Gly Gly Val Leu Val His
20 25 30
Pro Gln Trp Val Leu Thr Ala Ala His Cys Ile Arg Asn Lys Ser Val
35 40 45
Ile Leu Leu Gly Arg His Ser Leu Phe His Pro Glu Asp Thr Gly Gln
50 55 60
Val Phe Gln Val Ser His Ser Phe Pro His Pro Leu Tyr Asp Met Ser
65 70 75 BO
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Leu Leu Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His Asp
85 90 95
Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu Thr Asp Ala Val
100 105 110
Lys Val Met Asp Leu Pro Thr Gln Glu Pro Ala Leu Gly Thr Thr Cys
115 120 125
Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Glu Phe Leu Thr Pro
130 135 140
Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser Asn Asp Val Cys
145 150 155 160
Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala Gly
165 170 175
Arg Trp Thr Gly Gly Lys Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro
180 185 190
Leu Val Cys Asn Gly Val Leu Gln Gly Ile Thr Ser Trp Gly Ser Glu
195 200 205
Pro Cys Ala Leu Pro Glu Arg Pro Ser Leu Tyr Thr Lys Val Val His
210 215 220
Tyr Arg Lys Trp Ile Lys Asp Thr Ile Val Ala Asn Pro
225 230 235
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 237 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val
1 5 10 15
Ala Val Tyr Ser His Gly Trp Ala His Cys Gly Gly Val Leu Val His
20 25 30
Pro Gln Trp Val Leu Thr Ala Ala His Cys Leu Lys Lys Asn Ser Gln
35 40 45
Val Trp Leu Gly Arg His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gln
50 55 60
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b9
Arg Val Pro Val Ser His Ser Phe Pro His Pro Leu Tyr Asn Met Ser
65 70 75 BO
Leu Leu Lys His Gln Ser Leu Arg Pro Asp Glu Asp Ser Ser His Asp
85 90 95
Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Lys Ile Thr Asp Val Val
100 105 110
Lys Val Leu Gly Leu Pro Thr Gln Glu Pro Ala Leu Gly Thr Thr Cys
115 120 125
Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Glu Phe Leu Arg Pro
130 135 140
Arg Ser Leu Gln Cys Val Ser Leu His Leu Leu Ser Asn Asp Met Cys
145 150 155 160
Ala Arg Ala Tyr Ser Glu Lys Val Thr Glu Phe Met Leu Cys Ala Gly
165 170 175
Leu Trp Thr Gly Gly Lys Asp Thr Cys Gly Gly Asp Ser Gly Gly Pro
180 185 190
Leu Val Cys Asn Gly Val Leu Gln Gly Ile Thr Ser Trp Gly Pro Glu
195 200 205
Pro Cys Ala Leu Pro Glu Lys Pro Val Val Tyr Thr Lys Val Val His
210 215 220
Tyr Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala Asn Pro
225 230 235
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
ACGCGGATCC AGCATGTGGG ACCTGGTTCT CT 32
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
II
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
ACAGCTGCAG TTTACTAGAG GTAGGGGTGG GAC 33
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
{xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ATATGGATCC ATATGTCAGC ATGTGGGACC TGGTTCTCTC CA 42
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
ATATGGATCC TCAGGGGTTG GCTGCGATGG T 31
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
TCATCTCTGT ATCC 14
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
\
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
GAGTAAGCTC TA 12
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE; cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
GATGACTCCA GCCACGACCT 20
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
CACAGACACC CCATCCTATC 2O
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
GAGGGTTGTG TACAGTCATG GAT 23
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
II
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
ACACACTGAA GACTCCTGGG GCG 23
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 19 amino acids
(B} TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Glu Lys His Ser Gln Pro Trp Gln Val Ala Val Tyr Ser His Gly Trp
1 5 10 15
Ala His Cys
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B} TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
His Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:21:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
{C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
His Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys
1 5 10 15
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(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Ala Val Tyr Ser His Gly Trp Ala His Cys Gly Gly Val Leu Val His
1 5 10 15
Pro Gln Trp Val Leu Thr Ala Ala His Cys Leu Lys Lys Asn Ser Gln
20 25 3p
Val Trp Leu Gly Arg His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gln
35 40 45
Arg Val Pro Val Ser His Ser
50 55
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 711 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
ATTGTGGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGCTTGTGGCCTCT60
CGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCC120
CACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAGCCTGTTTCATCCTGAA180
GACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCGCTCTACGATATGAGC240
CTCCTGAAGAATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTC300
CGCCTGTCAGAGCCTGCCGAGCTCACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAG360
GAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCTGGGGCAGCATTGAACCAGAGGAG420
TTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAATGACGTGTGT480
GCGCAAGTTCACCCTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTGGACAGGG540
GGCAAAAGCACCTGCTCGGGTGATTCTGGGGGCCCACTTGTCTGTAATGGTGTGCTTCAA600
GGTATCACGTCATGGGGCAGTGAACCATGTGCCCTGCCCGAAAGGCCTTCCCTGTACACC660
AAGGTGGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACCCC 711
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
II
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(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE. peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu Phe Glu
1 5 10 15
Pro Glu Asp Thr Gly Gln Arg Val
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Cys Ala Leu Pro Glu Lys Pro Ala Val Tyr Thr Lys Val Val His Tyr
1 5 10 15
Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala
20 25
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Gln Val Ala Val Tyr Ser His Gly Trp Ala His Cys
1 5 10
